Akt1 players promote PMA U937 cell line differentiation into macrophage-like cells

Purpose – Monocytes are a leukocytes ’ subset that plays an important role in immunity. Protein kinase B (AKT) is involved in monocytes ’ survival, proliferation and differentiation. Using phorbol 12-myristate 13-acetate (PMA) as an inducer for cell line U937 differentiation into macrophage-like cells may be used as a modelforcancercelltherapyorotherbiomedicalresearchstudies.TheauthorsinvestigatedtheAkt1signaling pathway ’ s involvement with PMA as a differentiating agent and survival in the U937 cell line. Design/methodology/approach – PMAwasutilizedtostimulatethedifferentiationoftheU937celllineinto macrophage-like cells at a concentration of 10 nM. Akt1-phosphorylated Serine 473, Bad-phosphorylated Serine 136 and Caspase9-phosphorylated Serine 196 were tested by flow cytometry for the involvement of the Akt1 signaling pathway during differentiation in addition to the expression of CD14, CD206 and CD83. DNA cell cycle variation analysis was done using PI staining and cell viability and apoptosis detection using Annexin V and PI flow cytometry. Findings – There was a decrease in phosphorylated Akt1 and Bad activation and an increase in Caspase9 activation, with an increase in surface markers CD14, CD206 and CD83 acquired by PMA-differentiated cells. DNA cell cycle analysis revealed cell accumulation in the G2/M phase and fewer cells in the S phase of PMA-inducedU937.ApoptosisinductionforLy294002orWortmannin-inhibitedcellsandpartofPMA-inducedcellsweredetected. Originality/value – Theseresultsmaybeusedtocreateamodelforbiomedicalresearchstudiesandadvance the understanding of the mechanism involving differentiation of the U937 cell line.


Introduction
Monocytes are a type of leukocyte with critical roles in inflammation, pathogen challenge and homeostasis.These immune cells, produced by the myeloid progenitors in the bone marrow, account for about 10% of leukocytes in the bloodstream and ultimately migrate to peripheral tissues (van Furth & Sluiter, 1986).Monocytes are characterized by the high expression of CD14, a lipopolysaccharide-binding protein targeting endotoxins, and demonstrate extensive plasticity and heterogeneity in response to immunological challenges.Monocytes are programmed to undergo apoptosis within 24-48 h without specific survival signals (Cline, Lehrer, Territo, & Golde, 1978;Goyal et al., 2002) or get differentiated into either macrophages or antigen-presenting cells upon cell activation (Lu & Pitha, 2001).Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is involved in cell survival and proliferation (Brazil & Hemmings, 2001;Datta, Brunet, & Greenberg, 1999).AKT-1 is a 57 kDa serine/threonine protein kinase, phosphorylating and regulating the function of many cellular proteins involved in metabolism, survival, apoptosis, differentiation and proliferation (Brazil, Yang, & Hemmings, 2004).Thr308 and Ser473 are two distinct phosphorylation sites for Akt activation.Akt is activated by phospholipid binding (PIP3) and activation loop phosphorylation at threonine 308 by PDK1 (phosphoinositide-dependent protein kinase-1) (Dangelmaier et al., 2014), C-terminus phosphorylation at serine 473; this will lead to complete activation of Akt and could be achieved by one of the following: the PI3K-related kinase (PIKK) family, the mTOR/rictor complex (mTORC2), the DNA-dependent protein kinase (DNA-PK) (Fayard, Tintignac, Baudry, & Hemmings, 2005) and the TCR (Yang, Qiao, Ying, Zhang, & Yin, 2010).Constitutive AKT-1 activation is essential for the survival of monocyte differentiated macrophages (Liu, Perlman, Pagliari, & Pope, 2001) and involves downstream effectors such as Bad and Caspase-9 (Datta et al., 1997;Vincent & Feldman, 2002).Immortalized monocyte-like cells, U937 cells, are one of the cell lines mimicking human peripheral blood mononuclear cells (PBMC).The U937 cells were isolated from the histiocytic lymphoma of a 37-year-old male patient (Sundstrom & Nilsson, 1976).U937 cells, depending on the initiators, can adopt a monocytic phenotype and differentiate into either macrophages or dendritic cells.This investigation aimed to explore new signaling pathways during U937 cells' differentiation into macrophage-like cells by PMA, Phorbol 12-Myristate 13-Acetate.Here, we tested the AKT-1 signaling pathway and some of its downstream players, including Bad and Caspase9 activation involvement, in addition to the expression of regulatory cell surface markers CD14, CD206 and CD83.In the present study, we highlighted how AKT signaling appears as a vital regulator of differentiation, survival and surface expression of CD14, CD206 and CD83 in PMA-induced U937 cell differentiation to macrophage-like cells.Furthermore, PMA promoted U937 cell accumulation in the G2M phase of the cell cycle during differentiation.Our findings can provide a valuable model of the molecular mechanism of cell differentiation and survival as it relates to monocyte development.

Cells
U937 cells were purchased from the American Type Culture Collection (ATCC, No. CRL-1593.2,Virginia, USA).Cells were grown in RPMI-1640 medium (American Type Culture Collection, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco by life technologies corporation, NY, USA), 1% of Penicillin-streptomycin (Gibco by life technologies corporation, NY, USA) and incubated at 378C, 95% humidity, and 5% CO 2 .For cells viability and cells count, trypan blue execution method with 0.4% trypan blue stain using a hematocytometer was carried out.On average, viable cells more than 95% were used for the tests at a cell density of 1 3 10 6 cells/ml were used per each test.

Antibodies and reagents
All antibodies used for protein expression of CD14, CD206 and CD83, in addition to AKT phosphorylation and annexin V-FITC apoptosis detection kit, were obtained from BD Biosciences, NJ, USA.Antibodies for caspase9 pSer196 and bad pSer136 phosphorylation were from Santa Cruz Biotechnology, Texas, USA.Ly294002 and Wortmannin inhibitors were obtained from Cell Signaling Technology, Massachusetts, USA.PMA was from Abcam Biochemicals, Cambridge, UK.
U937 cell line activation and inhibition U937 cells were plated in 24-well plates (5 3 10 5 cells/ml/well).Ly294002 (50 mM) (Cell Signaling Technology) or Wortmannin (1 mM) (Cell Signaling Technology) were used to inactivate the AKT pathway for 1 hour at 37ºC before cell stimulation with PMA (10 nM) (Abcam Biochemicals).Control cells were either stimulated with PMA (10 nM) to induce macrophage differentiation or vehicle control.Cells were first incubated for 24 hours and used for protein phosphorylation detection studies or changed their media with fresh RPMI-1640 complete media, then incubation expanded to more than 48 hours at 378C, 95% humidity and 5% CO2 incubator to assess morphological changes, cell cycle analysis, annexin V apoptosis detection and cell surface marker expression.Cells pictures were taken using an Olympic light microscope with magnification using a 403 objective.

Cell surface marker expression detection assay by flow cytometry
Expression of CD14 (BD Biosciences, CD14 PerCP-Cy5.5);CD83 (BD Biosciences, CD83 APC); and CD206 (BD Biosciences, CD206 FITC) were measured following 72 h postactivation.Briefly, 20μl of each labeled antibody for every 100μl of cell suspension containing 1 3 10 6 cells was added and incubated at room temperature protected from light for 15 minutes.Cells were washed with 1X PBS and analyzed by flow cytometry.

DNA staining for cell cycle analysis by flow cytometry
Following U937 activation for 72h, cells were harvested, fixed and permeabilized by cold ethanol 70%.Cells were then washed with 1X PBS and stained with 2.5 mg/ml propidium iodide mixed with 0.5 mg/ml RNase A.

Annexin V apoptosis detection assay by flow cytometry
The Annexin V-FITC apoptosis detection kit (BD Biosciences) was used following manufacturer instructions.Cells were harvested, washed with cold 1X PBS and then resuspended in 1X binding buffer at a cell density of 1 3 10 6 cells/ml.100ml (1 3 10 5 cells) were stained with FITC Annexin V and PI stains followed by flow cytometry analysis.

Statistical assessment
For comparison between groups A student t-test was used.Statistically significant results were defined as p ≤ 0.05.

Akt1 players and cell differentiation
Results U937 cells show a macrophage-like morphology following PMA differentiation Microscopic analysis following 72 hours of PMA incubation, U937 cells developed pseudopods, a macrophage-like morphology, and adhesion, as shown in (Figure 1A and B).Dead cells and fragmented cells were observed in Wortmannin-treated cells (Figure 1C) and Ly294002 treated cells (Figure 1D).After treatment with PMA for 72 h, we detected a decrease in the number of cells when compared with untreated cells (Figure 1B).When cotreated with Wortmannin, an inhibitor of PI3 kinase or Ly294002 the number of cells decreased and differentiated cells.This indicates the AKT1 cell signaling pathway is essential for cell differentiation and survival.
AKT controls apoptosis and participates in differentiation after PMA in U937 cells Wortmannin or Ly294002 are potent inhibitors of the AKT signaling pathway.Cells were incubated for 1 hour with Wortmannin or Ly294002 before activation with PMA (10 nM) for 24 hours.Inhibition of AKT1 by Wortmannin or Ly294002 revealed a decrease in Akt-pSer473 phosphorylation, which triggered induction of apoptosis.Our annexin V and propidium iodide apoptosis detection by flow cytometry indicated a high percentage of apoptotic cells when inhibiting the AKT1 pathway (Figure 7).Following PMA treatment, Akt-pSer473 phosphorylation was also reduced, demonstrating the importance of AKT1 activity in the U937 differentiation process (Figure 2).Earlier research revealed a connection between AKT and PKC in PMA, specifically the activating or inhibiting actions of Akt1-pSer473.According to (Barragan et al., 2006), PMA activates AKT in B-CELL cells, B-cell chronic lymphocytic leukemia cells, without the need of PI3-K but rather through PKCβ leading to an increase in Akt1-pSer473 phosphorylation.On the other hand, according to Liu  (Liu, Qiu, Xiao, & Dong, 2006), PMA can specifically prevent G-CSF from activating A-pSer473 through PKC.PMA's ability to inhibit Akt during G-CSF-induced granulocytic differentiation is linked to a decrease in PKCᵋ expression.In our study, PMA led to a decrease in Akt1-pSer473, and this reduction led to the induction of differentiation of U937 cells into macrophage-like cells.The addition of Wortmannin or Ly294002, which are potent inhibitors of AKT, led to an increase in the reduction of Akt-pSer473 and led to apoptosis, which mainly indicates the activation is through the Akt pathway with potential PKC pathway involvement since according to Liu H. findings, PMA reduction of Akt1-pSer473 may be through PKC in previously activated Akt1 U937 proliferating cells.

Inhibition of the AKT pathway led to a reduction in bad phosphorylation
Inhibition of the AKT pathway before PMA U937 cell treatment for 24 h indicated a decrease in phospho-serine136 Bad.Also, PMA treatment of U937 cells for 24 h showed a decrease in phospho-serine136 Bad level (Figure 3) due to a decrease in activated AKT1 upon PMA stimulation.Bad is a proapoptotic protein regulated by phosphorylation of serine-122 and serine-136.Phosphorylation at either site can result in Bad losing its ability to heterodimerize with Bcl-xL or Bcl-2 survival proteins.Serine-136 phosphorylation was shown to be dependent on AKT activation (Datta et al., 1997).Caspases are intracellular cysteine-aspartic proteases that act as initiators and effectors of apoptosis, as well as have nonapoptotic roles associated with cell proliferation, differentiation, tumor suppression and others (Hart & Vogt, 2011).Caspase-9 is an initiator caspase influenced by the AKT signaling pathway.Caspase 9 can be phosphorylated at multiple sites by different protein kinases, leading to inhibition of caspase 9 activations (Shalini, Dorstyn, Dawar, & Kumar, 2015).The Serine 196 position is the position of activated AKT to inhibit caspase 9. Activated AKT1 induces phosphorylation of Caspase 9 at Ser196 and inhibits its protease activity, therefore inhibiting downstream apoptosis activation.Flow cytometry was performed to confirm the role of caspase-9 phosphorylation at serine 196 by activating AKT1 as the main player in U937 cell survival upon PMA activation.Preincubation with Ly294002 or Wortmannin of PMA-treated U937 cells reduced PMAinduced caspase 9 phosphorylation at serine 196 (Figure 4).

Expression of cell surface markers during U937 cell differentiation
A significant expression of CD14 membrane protein was detected by flow cytometry following the induction of U937 cells with PMA.However, the expression of CD206 and CD83 proteins increased slightly following PMA treatment.Increased CD83 expression on the surface of differentiated cells may confer antigen-presenting cell capability.The inhibition of the AKT pathway by Ly294002 or Wortmannin before cell PMA treatment showed a decrease in CD14, CD206 and CD83 surface marker expression (Figure 5).These findings highlight the role of the AKT1 pathway in the expression of proteins critical to macrophage activity.

Akt1 players and cell differentiation
PMA induces an increase in G2/M phase during the differentiation of U937 cells U937 cells treated with PMA for 72 hours showed a decrease in the number of cells found in the G1 and S phases and an accumulation in the G2/M phase of the cell cycle and viability test (Figure 6).

Akt1 players and cell differentiation
Discussion U937 cells are one of the cells that can be used as a model to study monocyte/macrophage functions.However, to be used as an in vitro model for macrophages, U937 cells must be differentiated, and several protocols have been tested (Allan & Clarke, 2009), (Prasad et al., 2020), (Scotti et al., 2018).PMA is known as one of the agents that led to the differentiation of U937 cells into macrophage-like cells and other cells such as MO3.13 cells (Damato et al., 2021), HOP 92 cells (Choi, Hyman, & Blumberg, 2006).
The differentiation mechanisms that lead to the PMA-induced U937 differentiation are governed mainly by the balanced activation of kinases and phosphatases.Several pathways are involved, such as the activation of the MAPK/ERK pathway (Olsson, Gullberg, Ivhed, & Nilsson, 1983) with different stimuli, including SET, a nuclear phosphoprotein, which leads to dendritic-like differentiation of U937 cells (Olsson et al., 1983).RhoA/ROCK signaling, induced by PMA (100 ng/ml), was found to be associated with U937 cell-line into differentiation and characterized by the expression of CD68 (Kandilci & Grosveld, 2005).Our study investigates the early involvement of AKT pathway activation in PMA-induced differentiation of U937 cells.One of the significant findings of this study is the hypophosphorylation of AKT1 in U937 cells following PMA activation for 24 hours, which leads to the differentiation by suppression of cell proliferation.The reduced AKT1phosphorylation plays a role in differentiated cell survival by the reduction of Bad phosphorylation, an Akt-downstream survival mediator.In addition, PMA treatment of U937 A study in 2021 demonstrated an increase of Caspase 9 activity during differentiation (Madadi, Akbari-Birgani, Mohammadi, Khademy, & Mousavi, 2021).Inhibition of AKT1 activity by Ly294002 or Wortmannin significantly decreases Bad and Caspase 9 phosphorylation, promoting apoptosis.
Previous studies have indicated that the AKT pathway is necessary for cell survival and differentiation (Yang, Dai, Tang, Le, & Yao, 2017;Abdullah, Hills, Winter, & Huang, 2021).Our results show that AKT1 activity is reduced as PMA induced-U973 differentiation.In addition, Bad phosphorylation reduction, as a consequence of the PMA effect on AKT1 phosphorylation profile, may not be necessary for cell survival; rather, it may be obligatory for U937 cell differentiation induction.PMA suppresses Akt1-PSer473 phosphorylation, possibly via PDK1, and because Bad is downstream of Akt1, as a result, it lessens the downstream activation of Bad pSer136 (Figure 8).In addition, our results showed that cell adhesion in PMA-treated cells led to differentiation compared with untreated rounded cells.In this study, we demonstrated the ability of 10 nM PMA to induce an increase in the expression of cell surface markers such as CD14, CD206 and CD83.The

Akt1 players and cell differentiation
In our study, a slight increase in CD83 expression was found in U937-PMA stimulated cells.CD83, a member of the immunoglobulin superfamily of receptors associated with antigen presentation, is expressed by monocytes, macrophages, and dendritic cells.CD83 expression is transient on activated monocytes and macrophages but stable on activated dendritic cells (Cao, Lee, & Lu, 2005).The slight increase in CD83 expression may allow these cells to gain antigen-presenting cell capabilities (Cao et al., 2005).An increase in the expression of CD206 in PMA-differentiated U937 cells was observed in our study.Macrophages express various scavenger receptors, such as CD206, which are upregulated in response to inflammation (Nielsen et al., 2020).CD206 is a 175kDa membrane-bound protein, primarily expressed by macrophages and dendritic cells.CD206 is involved in endogenous molecule clearance, antigen presentation and modulation of cellular activity.In addition, CD206 is considered an efficient endocytic receptor that continuously recycles between the cell surface and early endosomal compartments (Gazi & Martinez-Pomares, 2009).Although we did not examine the phagocytic activity of U937 differentiated cells, we provide evidence of differential expression of the protein.Our cell cycle study indicated an accumulation of U937-PMAtreated cells in the G2/M phase with a concomitant reduction in the S and G1 phases after 72 hours.Exit from the cell cycle by G1-phase arrest is one of the indications of U937 cell differentiation (Asada, Yamada, Fukumuro, & Mizutani, 1998).However, others have reported G2/M arrest in differentiated U937 cells (Devlin et al., 2003).Apoptosis was estimated by annexin V-FITC (Nagata, Suzuki, Segawa, & Fujii, 2016) and demonstrated that PMA treatment of U937 cells, 23% differentiated cells and 77% were apoptotic cells.PMA combined with Ly294002 U937 cell treatment increased the number of apoptotic cells, indicating the AKT's importance for cell survival.
Figure 1.Morphology of U937 Cells Figure 2. AKT1 participates in the induction of differentiation by PMA in U937 cells Figure 3. PMA decreases phosphorylation of Ser136 on Bad via AKT in U937 cells Figure 4. PMA upsurges Caspase9 phosphorylation on Ser196 in U937 cells Figure 5. Surface marker expression in U937 cells following PMAinduced differentiation Figure 6.PMA induces an increase in the G2/M phase during the differentiation of U937 cells Figure 7. Apoptosis detection inhibition of the AKT1 pathway by Ly294002 decreased the expression of CD14, CD83 and CD206.Previous research has shown that CD14 expression is dependent on the induction of a protein intermediate, SP1, which is induced by [1,25(OH)2D3] (Busca, Saxena, Iqbal, Angel, & Kumar, 2014).In addition, Baek et al. reported that retinoic acid could increase the expression of CD14 in U937 cells following exposure to [1, 25(OH)2D3] (Zamani, Zare Shahneh, Aghebati-Maleki,& Baradaran, 2013).Despite the fact that previous studies of PMA-induced U937 differentiation failed to induce CD14 expression(Baek et al., 2009), our study's indication of CD14 expression may be due to the use of different doses of 10 nM PMA.
Figure 8. Diagram showing potential AKT involvement in the differentiation of PMA-U937 cells