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AKT-mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells

  • Original Research Paper
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Abstract

Objectives

Neutrophil polarization is critical for the inflammatory response. AKT is a serine/threonine protein kinase and has been implicated in cell migration. However, it is not completely clear whether AKT affects neutrophil polarization. In this study, we tested the hypothesis that AKT regulates the polarization of neutrophil-like differentiated HL-60 cells (dHL-60) in response to fMLP.

Methods

HL-60 cells were differentiated into dHL-60 by incubation in medium containing 1.3 % DMSO for up to 6 days. Polarization of dHL-60 cells and primary human neutrophils were measured by Zigmond chamber. Phospho-Akt was analyzed by immunofluorescence and Western blot analysis. F-actin polymerization was detected by Rhodamine-Phalloidine staining. Rac2 activation was evaluated using GST Pull-down assay.

Results

We found that changes in the rate of cell polarization were consistent with the changes in AKT phosphorylation levels during HL-60 cell differentiation in response to fMLP. Moreover, cell polarization and AKT phosphorylation were reduced in fMLP-stimulated dHL-60 cells pretreated with the PI3 kinase inhibitors or the AKT inhibitors, which was confirmed in the primary human neutrophils. The AKT inhibitors altered fMLP-induced F-actin polymerization. Rac2 GTPases was also decreased by the AKT inhibitors in fMLP-stimulated dHL-60 cells.

Conclusion

This study demonstrates that AKT activation plays a crucial role in dHL-60 cell polarization.

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Abbreviations

dHL-60:

Differentiated HL-60

DMSO:

Dimethylsulfoxide

fMLP:

N-Formyl-Met-Leu-Phe

PBS:

Phosphate-buffered saline

PH domain:

Pleckstrin homology domain

PI3K:

Phosphoinositide 3-kinase

PIP3:

Phosphatidylinositol (3,4,5)-trisphosphate

TORC2:

Target of rapamycin complex 2

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Acknowledgments

This work was supported by grants from National Basic Research Program of China, No. 2012CB518200 (FZ) and No. 2012CB525004 (XM), National Natural Science Foundation of China No. 30971193 (FZ), No. 30800438 (XM) and No. 81071611 (CC).

Conflict of interest

The authors declare that there are no conflicts of interest.

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Authors and Affiliations

  1. Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China

    Wenying Zou, Xinwei Chu, Chunqing Cai, Xiaojing Meng, Haiyang Chen & Fei Zou

  2. Department of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical University, Guangzhou, China

    Mengchen Zou

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  1. Wenying Zou
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  2. Xinwei Chu
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  3. Chunqing Cai
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  7. Fei Zou
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Correspondence to Fei Zou.

Additional information

Responsible Editor: Liwu Li.

W. Zou, X. Chu and C. Cai equally contributed to this paper.

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Zou, W., Chu, X., Cai, C. et al. AKT-mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells. Inflamm. Res. 61, 853–862 (2012). https://doi.org/10.1007/s00011-012-0478-y

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  • DOI: https://doi.org/10.1007/s00011-012-0478-y

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