Mechanism Of Autophagy In ATRA-induced Myeloid Cell Differentiation

Acute promyelocytic leukemia (APL) is a common variant of acute myeloid leukemia (AML). The PML-RAR?oncogene [a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-?(RAR?)] is expressed by the acute promyelocytic leukemia. All trans retinoic acid (ATRA) induces degradation of the fusion protein encoded by the PML-RAR?oncogene, differentiation of leukaemic cells and clinical remissions. It is not clear to present scientists yet about the mechanism and significance of autophage regulating degradation of PML/RAR?oncoprotein.In this paper, we first use immunofluoresence, Western blotting, flow cytometric analysis and transmission electron microscopy analysis to observe the ATRA- induced autophagy in myeloid cells. We found ATRA at therapeutic doses induced autophagy in myeloid cells; in contrast, the autophagy was blocked with the addition of an autophagy inhibitor,3-methyladenine (3-MA). Moreover, we explored the significance of autophagy-related gene (ATG) in the process of ATRA-induced autophagy. Suppression of ATG in myeloid cells by RNA interference resulted in the inhibition of ATRA induced autophagy, and rendered them significantly more sensitive to ATRA-induced apoptotic cell death.At second part of this paper, in order to determine the impact of autophagy on myeloid differentiation, HL-60 and NB4 cells were incubated with anautophagy inhibitor (e.g.3-MA) or an autophagy inducer (e.g. rapamycin) in the presence or absence of ATRA, followed by flow cytometric analysis of the myeloid differentiation marker CD11b. The autophagy inhibitor decreased the cell differentiation; to the contrary, the autophagy inducer increased it. Moreover, knockdown of Atg5 by RNA interference(RNAi) impaired the capacity of other differentiating agents to induce differentiation of myeloid cells.Finally, we found that Autophagy regulates ATRA-induced degradation of PML-RAR?in human myeloid cells; The interaction between p62 and PML-RAR?regulates degradation of PML-RAR?and myeloid cell differentiation. To determine the mechanism by which autophagy alters myeloid cell differentiation, the degradation of PML/RARa was decreased when degradation was blocked by the proteasome, caspases or autophay. knockdown of Atg5 by RNAi decreased degradation of PMLRAR?and the autophagy. To explore whether p62 binds to PMLRAR?, we performed co-immunoprecipitation (Co-IP) analysis using p62 and RAR?antibodies. Consistently, the colocalization between PML-RAR?and p62 by immunofluoresence were increased after ATRA treatment, confirming an interaction between p62 and PML-RAR?. Suppression of p62 by RNAi inhibited the cell differentiation and the degradation of PML/RAR?; whereas enhanced expression of p62 by gene transfection increased the delivery of PML-RAR?proteins to the lysosome. These studies suggest that appropriate levels of p62 contribute to sustained PML-RAR?levels during cell differentiation by the coordination of proteolytic crosstalk between the proteasomal and autophagic pathways.In conclusion, ATRA inhibits the mTOR pathway and actives the Atg1-PI3KC3-Atg5 dependent autophagy pathway, which promotes autophagosomes formation. Furthermore, the interaction between p62 and PML-RARa regulates degradation of PML-RAR?. Inhibition of p62 impaired the degradation of PML-RAR?. In contrast, inhibition of autophagy results in an accumulation of p62, which through a negative feedback mechanism inhibits the activity of the proteasome as well as degradation of PML-RAR?. Thus, autophagy plays an important role in regulating degradation of the PML-RAR?oncoprotein and myeloid cell differentiation by p62 during cell differentiation.