Elsevier

Nitric Oxide

Volumes 109–110, 1 May 2021, Pages 33-41
Nitric Oxide

Phorbol myristate acetate induces differentiation of THP-1 cells in a nitric oxide-dependent manner

https://doi.org/10.1016/j.niox.2021.02.002Get rights and content

Highlights

  • Phorbol myristate acetate induces monocyte-to-macrophage differentiation ofTHP-1 cells.

  • Differentiation of THP-1 cell partially depends on inducible nitric oxide synthase-triggered nitric oxide.

  • Calcium signaling may mediate upregulation of nitric oxide by phorbol myristate acetate.

Abstract

Introduction

THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Here we elucidated whether NO relates to PMA-induced monocyte-to-macrophage differentiation of THP-1 cells. The mutual regulation of calcium and NO was also investigated.

Material & methods

THP-1 cells were incubated with PMA for 24 h, followed by assay of adherence, morphological change, migration or IL-1β release. L-NG-Nitroarginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) or BAPTA-AM (a calcium chelator) was added before PMA stimulation, and levels of calcium and NO were measured. Furthermore, a selective inhibitor of inducible nitric oxide synthase (iNOS) activity was employed to study the role of iNOS.

Results and Discussion

Effects of PMA on upregulation of adherence, lipopolysaccharide-triggered IL-1β, and migration ability of THP-1 cells were consistent with NO concentrations. Both l-NAME and BAPTA-AM mitigated effects of PMA on THP-1 cells differentiation. BAPTA-AM decreased levels of NO, while l-NAME had no effect on calcium levels. Of note, inhibition of iNOS activity decreased PMA-triggered upregulation of NO.

Conclusion

PMA induced differentiation of THP-1 cells partially in a NO-dependent manner. The calcium signaling may mediate PMA-triggered upregulation of NO.

Introduction

Monocytes and macrophages are both key immune effector cells [1] and regulate immunity through a variety of ways [2,3]. Monocyte-to-macrophage differentiation plays a critical role in many inflammatory diseases, such as atherosclerosis [[4], [5], [6]]. During progress of atherosclerosis, monocytes migrate into subendothelial spaces where they differentiate to macrophages [4,7,8]. Macrophages accumulate in atherosclerotic plaques promoting disease exacerbation [4,7,8]. In this regard, some therapeutic drugs for atherosclerosis target inhibition of monocyte-to-macrophage differentiation or macrophage activities [9,10].

The biology and function of macrophages are widely studied in many in vitro researches. Differentiated THP-1 cells, a human leukemia monocytic cell line, are widely used as surrogate of human macrophages and serve as a reliable cellular model [11,12]. THP-1 cells can be differentiated into macrophages by phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator [13,14]. After differentiated with PMA, THP-1 cells acquire macrophage-like characteristics [14,15]. Increased cell adherence and decreased proliferation rate are found in PMA-differentiated THP-1 cells [[14], [15], [16]]. PMA also enhanced lipopolysaccharide (LPS)-triggered release of pro-inflammatory cytokines [15], and upregulates migration ability of THP-1 cells [17,18].

Nitric oxide (NO) is a crucial intracellular messenger modulating various cellular signaling pathways [19]. The production of NO is generated by nitric oxide synthases (NOS), including inducible nitric oxide synthase (iNOS) [20] primarily found in a variety of immune cells [21]. From previous reports, immune cell activities can be regulated by NO signaling [[22], [23], [24]]. For example, NO regulates differentiation of dendritic cells [23] as well as lymphocyte migration capacity [24]. Though PMA stimulation upregulates NO and iNOS [25,26], whether effects of PMA on THP-1 cells differentiation relate to NO or iNOS remain unstudied.

In this study, we sought to clarify whether the mechanism underlying PMA-induced monocyte-to-macrophage differentiation of THP-1 cells relates to NO or iNOS. Based on previous report, PMA triggers upregulation of calcium levels [27]. Furthermore, there is crosstalk between NO and calcium signaling [28]. For example, NO regulates intracellular calcium levels mediated by acting on NO receptors [29]. On the other hand, calcium-mediated signaling regulates iNOS [30,31]. Therefore, the effect of PMA on mutual regulation between calcium and NO was also investigated in this study.

Section snippets

Cell culture and stimulation

THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were grown in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). Cells of fewer than 15 passages were incubated in a humidified chamber at 37 °C in a mixture of 95% air and 5% CO2, and employed in this experiment. To facilitate investigation, THP-1 cells were incubated with or without 0-to-200 nM PMA for 24 h

Cell viability was not reduced by PMA up to 200 nM

Fig. 1 illustrated the data of cell viability measured through LDH release assay (n = 5 in each group). The cell viability in groups treated with 50-to-200 nM PMA was not different from that in the PBS group.

PMA increased cell adherence in a dose-dependent manner

The cell adherence was measured and shown in Fig. 2A (n = 5 in each group). Our data revealed that cell adherence in groups treated with PMA all increased significantly compared to the PBS group (all P < 0.001). Furthermore, there was significant difference between groups treated with PMA

Discussion

Our data showed that PMA induces differentiation of THP-1 cells in a NO-dependent manner. The correlation between NO levels and PMA-induced effects was obvious under dose of PMA between 50 nM and 200 nM. Though our results (Fig. 7B) showed l-NAME deceased the NO concentration in the PMA 200 nM group (P = 0.008), level of NO in the PL group was still significantly higher than the PBS group (P < 0.001). Therefore, we may suggest PMA-induced THP-1 cell differentiation partially depends on NO

Author contributions

YYC and TYL contributed to the conception and design, data collection, analysis and interpretation, writing and critical revision of the article. WHJ and CWL contributed to data analysis and interpretation as well as writing of the article. SJS contributed to critical revision of the article.

Acknowledgement

This work was supported by a grant from Far Eastern Memorial Hospital (FEMH-2020-C-022), awarded to YYC.

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