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Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
MAP kinase-dependent autophagy controls phorbol myristate acetate-induced macrophage differentiation of HL-60
Milos Mandic1, Maja Misirkic Marjanovic2, Ljubica Vucicevic2
1Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia.
Abstract:
We investigated the mechanisms and the role of autophagy in the differentiation of HL-60 human acute myeloid leukemia cells induced by protein kinase C (PKC) activator phorbol myristate acetate (PMA). PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by cell-cycle arrest accompanied by elevated expression of macrophage markers CD11b, CD13, CD14, CD45, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase in intracellular acidification, accumulation/punctuation of autophagosome marker LC3-II, and the increase in autophagic flux. PMA also increased nuclear translocation of autophagy transcription factors TFEB, FOXO1, and FOXO3, as well as the expression of several autophagy-related (ATG) genes in HL-60 cells. PMA failed to activate autophagy inducer AMP-activated protein kinase (AMPK) and inhibit autophagy suppressor mechanistic target of rapamycin complex 1 (mTORC1). On the other hand, it readily stimulated the phosphorylation of mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) via a protein kinase C-dependent mechanism. Pharmacological or genetic inhibition of ERK or JNK suppressed PMA-triggered nuclear translocation of TFEB and FOXO1/3, ATG expression, dissociation of pro-autophagic beclin-1 from its inhibitor BCL2, autophagy induction, and differentiation of HL-60 cells into macrophage-like cells. Pharmacological or genetic inhibition of autophagy also blocked PMA-induced macrophage differentiation of HL-60 cells. Therefore, MAP kinases ERK and JNK control PMA-induced macrophage differentiation of HL-60 leukemia cells through AMPK/mTORC1-independent, TFEB/FOXO-mediated transcriptional and beclin-1-dependent post-translational activation of autophagy.
Insights
Phorbol myristate acetate (PMA) induces differentiation of acute myeloid leukemia cells by activating autophagy. This process is mediated by mitogen-activated protein kinases (MAPK) ERK and JNK, independent of AMPK/mTORC1 signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Acute myeloid leukemia (AML) is a heterogeneous cancer with complex differentiation pathways.
- Autophagy, a cellular degradation process, plays a multifaceted role in cancer, including differentiation.
- Protein kinase C (PKC) activators like phorbol myristate acetate (PMA) can induce differentiation in leukemia cell lines.
Purpose of the Study:
- To elucidate the mechanisms underlying PMA-induced differentiation of HL-60 leukemia cells.
- To investigate the specific role of autophagy in this differentiation process.
- To identify the key signaling pathways involved in PMA-induced autophagy and differentiation.
Main Methods:
- HL-60 cells were treated with PMA to induce differentiation.
- Autophagy was assessed by measuring intracellular acidification, LC3-II accumulation, autophagic flux, and nuclear translocation of TFEB, FOXO1, and FOXO3.
- The involvement of MAP kinases (ERK, JNK), AMPK, mTORC1, and autophagy-related genes (ATG) was examined using pharmacological and genetic inhibition.
Main Results:
- PMA induced HL-60 cell differentiation into macrophage-like cells, confirmed by cell-cycle arrest and increased macrophage markers.
- PMA triggered autophagy, evidenced by increased autophagic flux and LC3-II punctation, and nuclear translocation of TFEB and FOXO transcription factors.
- PMA activated ERK and JNK signaling, which were essential for TFEB/FOXO nuclear translocation, ATG gene expression, beclin-1 activation, autophagy, and subsequent differentiation, independently of AMPK/mTORC1.
Conclusions:
- Mitogen-activated protein kinases ERK and JNK are critical regulators of PMA-induced macrophage differentiation in HL-60 leukemia cells.
- These MAP kinases mediate differentiation by activating autophagy through TFEB/FOXO-dependent transcriptional regulation and beclin-1-dependent post-translational mechanisms.
- The identified pathway is independent of the AMPK/mTORC1 signaling axis, highlighting a distinct mechanism for regulating leukemia cell differentiation via autophagy.
