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. Author manuscript; available in PMC: 2014 Jul 2.
Published in final edited form as: Chem Biol. 2012 Aug 24;19(8):994–1000. doi: 10.1016/j.chembiol.2012.06.010

Euphohelioscopin A is a PKC activator capable of inducing macrophage differentiation

Lorenzo de Lichtervelde 1, Corina E Antal 2,3, Anthony E Boitano 4, Ying Wang 5, Philipp Krastel 5, Frank Petersen 5, Alexandra C Newton 2, Michael P Cooke 4, Peter G Schultz 1,*
PMCID: PMC4079670  NIHMSID: NIHMS591025  PMID: 22921066

SUMMARY

To identify small molecules that selectively control hematopoietic stem cell (HSC) differentiation, we performed an unbiased screen using primary human CD34+ cells. We identified a plant-derived natural product, euphohelioscopin A, capable of selectively differentiating CD34+ cells down the granulocyte/monocytic lineage. Euphohelioscopin A also inhibits proliferation and induces differentiation of the myeloid leukemia cell lines THP-1 and HL-60. Mechanistic studies revealed that euphohelioscopin A is an activator of protein kinase C (PKC), and that the pro-monocytic effects of this natural product are mediated by PKC activation. In addition to shedding new insights into normal hematopoiesis, this work may ultimately facilitate the application of stem cell therapies to a host of myeloid dysfunctions.

INTRODUCTION

Among the best characterized adult stem cells are hematopoietic stem cells (HSC), which can self-renew and differentiate into all blood lineages. At each step in this process, cells lose some of the differentiation and proliferation potential of their upstream progenitor. This phenomenon can be harnessed as a strategy against blood malignancies and leukemias which arise from the oncogenic transformation of a progenitor rather than a fully differentiated cell. In those cases, forcing the malignant progenitors to differentiate can put an end to their invasive proliferation (Leszczyniecka et al., 2001; Nowak et al., 2009),(Petrie et al., 2009). In contrast, numerous conditions lead to the depletion of specific blood lineages, either as a direct result of bone marrow failure or as a consequence of leukemia, viral infection or even aggressive treatments including radiation and chemotherapy. The ability to generate sufficient quantities of differentiated, functional cells to replenish the deficient compartment has long been a focus of regenerative medicine. To identify new pharmacological agents that selectively differentiate hematopoietic progenitors toward a desired effector cell, we carried out an unbiased cell-based screen with primary human CD34+ cells. The plant natural product euphohelioscopin A was identified which induces differentiation of HSCs, THP-1 and HL-60 cells to the granulocyte/monocyte (GM) lineage by activation of PKC.

RESULTS

Discovery of an HSC-differentiating natural product

To identify molecules that modulate the differentiation of HSCs, a library of 704 pure microbial and plant natural products was screened in vitro using primary human CD34+ cells isolated from mobilized peripheral blood. Cells were seeded into 384-well plates (2500 cells per well) in medium optimized for their self-renewal, yet permissive to myeloerythroid differentiation (serum free medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), flt3 ligand (Flt-3L), and interleukin-6 (IL-6)) (Boitano et al., 2010; Murray et al., 1999). Compounds were added at a final concentration of 1µM and the cells were incubated for 7 days, at which point the cultures, consisting of a mixture of CD34+ and differentiated cells, were analyzed by flow cytometry. The number of cells of each lineage and the number of remaining progenitors in the culture were determined based on cell surface phenotype: CD34+ (HSCs and progenitors), CD34- (lineage-committed cells) CD45ra+ (GM lineage), and CD45ra- (megakaryocyte/erythrocyte lineage). Using this assay, we observed that euphohelioscopin A, a lathyrane diterpene natural product first isolated in 1983 (Shizuri et al., 1983) (Figure 1), strongly induced the differentiation of CD34+ cells towards the GM lineage (CD34-/CD45ra+).

Figure 1.

Figure 1

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Structure of euphohelioscopin A.

Euphohelioscopin A induces differentiation to the GM lineage

Euphohelioscopin A decreased the percentage of CD34+ cells after 7 days from 29% in the vehicle treated cultures (DMSO 0.04%) to less than 13%, while the total number of viable nucleated cells (TNC) doubled compared to vehicle treated cultures (Figure 2A, B). Concurrently, CD45ra+ cells increased from about 50% of the vehicle treated cultures to 79% of the total population when treated with euphohelioscopin A, suggesting that the compound promotes selective differentiation of CD34+ cells towards the myeloid lineage (EC50 = 600nM). This was further supported by the observation that CD34-/CD45ra+ cells make up to 71% of the final cell population after treatment with compound for 7 days. The change in phenotype and TNC resulted in no net change in the number of CD34+ cells compared to vehicle, while the number of CD34-/CD45ra+ increased 4-fold. Finally, CD34+ cells treated with euphohelioscopin A for 7 days generated 10-fold more cells positive for CD11b, an integrin characteristic of monocytes and macrophages, than the vehicle. Together these results suggest that euphohelioscopin A selectively promotes terminal differentiation of hematopoietic stem and progenitor cells toward the myeloid line-age.

Figure 2.

Figure 2

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Euphohelioscopin A induces the differentiation of CD34+ and THP-1 cells. Panels A-C characterize the population of cells generated by primary human mobilized peripheral blood-derived CD34+ cells cultivated for 7 days, with vehicle (DMSO 0.04%), euphohelioscopin A (600 nM) or PMA (0.6 nM). (A) CD34 and CD45ra expression, analyzed by flow cytometry. (B) Fold-change in TNC (Inline graphic), number of CD34+ cells (Inline graphic), and number of CD34-/CD45ra+ cells (Inline graphic), relative to vehicle. (C) Colony forming potential of 80 CD34+ cells following 7 days of culture with vehicle or euphohelioscopin A: GEMM (Inline graphic), E (Inline graphic), G (Inline graphic), GM (Inline graphic), M (Inline graphic). (D) THP-1 cells were seeded in a 96-well plate (5000 cells per well) in RPMI+10% FBS, and treated with vehicle (0.01% DMSO) or euphohelioscopin A (1µM). TNC (DMSO: Inline graphic, eupho.A: Inline graphic) and CD11b expression (DMSO: Inline graphic, eupho.A: Inline graphic) were quantified by flow cytometry after the indicated number of days in culture. Data are represented as mean ± standard deviation.

A key characteristic of stem and progenitor cells is the ability to form colonies when plated in semi-solid media. The composition of the colonies reflects the lineage potential of the expanded cells. The different colonies include macrophage (M), erythroid (E), mixed macrophage/granulocyte (GM), and mixed GM, E and megakaryocyte (GEMM) and represent a stage of hematopoietic differentiation between HSCs and more terminally differentiated cells. The number of colonies obtained is linearly proportional to the colony-forming cell content of the input cell suspension where colonies containing cells of two or more lineages (mixed colonies) arise from an earlier progenitor than those containing cells of a single lineage. To confirm the differentiating effect of euphohelioscopin A, we examined the colony forming potential of CD34+ cells expanded with vehicle or euphohelioscopin A (2.5µM) for 7 days. Euphohelioscopin A treated cells showed a 3-fold decrease in total colony forming unit (CFU) potential compared to control cultures (Figure 2C), suggesting that the cells had differentiated and the cultures had decreased numbers of progenitors. Interestingly, GM colonies were the only colonies that did not experience a significant decrease in numbers; all other colonies decreased by more than 4-fold. These results are consistent with the differentiated phenotype of the 7-day cultured cells and support the hypothesis that euphohelioscopin A treatment of CD34+ cells selectively leads to an increase in non-proliferating, lineage-restricted myeloid cells by driving the differentiation of CD34+ progenitor cells rather than inducing lineage-restricted CD34-/CD45ra+ cells to proliferate.

To determine whether euphohelioscopin-A can induce the differentiation of leukemic cells with myeloid potential, we tested its activity on two myeloid leukemia cell lines, HL-60 and THP-1. After 4 days of culture with euphohelioscopin A (1µM), 50% of THP-1 cells were CD11b+ compared to less than 5% of vehicle treated cells, and the total number of cells was 2.6-fold lower than in the control, indicating that the inhibition of proliferation was concurrent with differentiation to mature macrophages (Figure 2D). A similar effect was observed with HL-60 cells: after 4 days of treatment, 26% of cells were CD11b+ (compared to less than 5% of control cells), and the total cell number was decreased 8-fold (Figure S1).

Euphohelioscopin A is a PKC activator

The only biological activity reported to date for euphohelioscopin A is inhibition of the drug transporter P-glycoprotein, as measured in a mitoxantrone efflux assay (Barile et al., 2008). However, this compound was significantly less effective than related diterpenes (IC50: 14µM, with maximal inhibition at 100µM), suggesting that this mechanism does not account for the molecule’s differentiation potential. A known pathway for selective macrophage differentiation is through the activation of PKC (Blumberg, 1988). A comparison of the differentiation activities of euphohelioscopin A with known PKC activators revealed that phorbol esters such as phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu) and mezerein afforded a very similar phenotype in the CD34+ cell differentiation assay (Figure 2A, B) as well as in their ability to differentiate and halt the proliferation of myeloid leukemia cell lines HL-60 and THP-1. Furthermore, PKC inhibitors were able to inhibit euphohelioscopin A-induced differentiation: addition of the PKC inhibitor Gö6983 (500nM) shifted the EC50 of euphohelioscopin A in the CD34+ differentiation assay (from 590nM to 2µM, Figure 3A), in the THP-1 differentiation assay (from 200nM to 850nM, Figure 3B), and in the HL-60 differentiation assay (from 1.7µM to 14.5µM, Figure S1). Euphohelioscopin A was also able to induce the phosphorylation of known downstream targets of PKC including ERK1/2 and MARCKS (Figure 4A). These results suggest that the pro-monocytic effects of euphohelioscopin A are mediated through PKC activation.

Figure 3.

Figure 3

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The differentiating activity of euphohelioscopin A is mediated by PKC. (A) Human mobilized peripheral blood CD34+ cells were seeded in 96-well plates (5000 cells per well) and treated with vehicle (DMSO) or euphohelioscopin A at the indicated doses, with or without the PKC inhibitor Gö6983 (500nM). Cell number and CD45ra expression were measured by flow cytometry after 7 days of culture. (B) THP-1 cells were seeded in 96-well plates (5000 cells per well) and treated with vehicle (DMSO) or euphohelioscopin A at the indicated doses, with or without the PKC inhibitor Gö6983 (500nM). Cell number and CD11b expression were measured by flow cytometry after 4 days of culture. Data are represented as mean ± standard deviation. Legend: DMSO (Inline graphic), DMSO + Gö6983 (Inline graphic), euphohelioscopin A (Inline graphic), euphohelioscopin A + Gö6983 (Inline graphic). See also Figure S1.

Figure 4.

Figure 4

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Euphohelioscopin A is a PKC activator. (A) HEK293T cells were treated for 10 min with vehicle, euphohelioscopin A or PMA at the indicated concentrations, and Western blots were performed for phosphorylation of downstream targets of PKC. Data is representative of 3 independent experiments. (B) HeLa cells transiently transfected with a PKC-specific FRET probe (CKAR) and rat PKC βII-RFP were imaged to measure PKC activation in real time upon stimulation with euphohelioscopin A or PMA. Base-line images were acquired for 3 minutes before addition of euphohelioscopin A or PMA. The PKC inhibitor Gö6983 was added 35 min after the addition of euphohelioscopin A or PMA. FRET ratios were measured by epifluorescence microscopy at 15 second intervals and normalized to the baseline, the average FRET ratio measured before compound addition. The normalized average FRET ratio is the average of these corrected values ± S.E.

To further corroborate this hypothesis we performed a real-time FRET-based PKC activity assay using CKAR, a reporter for PKC-mediated phosphorylation developed by Violin et al. (Violin et al., 2003). CKAR is a genetically encoded, fluorescent PKC-specific substrate that undergoes a conformational change when phosphorylated, which can be monitored by intramolecular FRET. Euphohelioscopin A (30µM) was able to induce a robust increase in PKC activity in HeLa cells cotransfected with CKAR and rat PKC-βII-RFP; the observed activation was reversed by the PKC inhibitor Gö6983 (Figure 4B). Euphohelioscopin A had no effect on CKAR T/A, a reporter lacking the phospho-acceptor site. These results confirm that euphohelioscopin A acts as a PKC activator.

DISCUSSION

In summary, we have identified the natural product euphohelioscopin A as a PKC activator and inducer of myeloid differentiation. Due to its pivotal role in pathways governing cell proliferation and differentiation, PKC has been a focus of interest as a pharmacological target for cancer therapy for the last 3 decades. In particular, several PKC-activating compounds have been investigated for their ability to induce differentiation of malignant myeloid progenitors (Hampson et al., 2005; Han et al., 1998b) or to alleviate chemotherapy-induced leukopenia (Han et al., 1998a). Though phorbol esters were originally the most promising candidates due to their potency, the need to balance their benefits and their tumor-promoting effects severely limited the scope of potential therapeutic applications. This led to the development of alternative PKC activating natural products such as bryostatins and ingenols, which differed in their isozyme specificity, their kinetics of PKC activation and their induction of PKC translocation to various subcellular compartments (Clamp and Jayson, 2002; Hampson et al., 2005; Hampson et al., 2010; Kedei et al., 2004).

As more PKC ligands came to be known, it became clear that, whether in vitro or in vivo, the spectrum of PKC-mediated responses was very broad. This revived the interest in finding molecules able to activate PKC with distinct activities and specificities. More recently, as structural details emerged on the mechanism of PKC activation, several attempts to rationally design PKC activating or inhibiting ligands have been described (Baba et al., 2004; Boije af Gennas et al., 2009; Keck et al., 2010; Kiriazis et al., 2011). In this report we introduce a new scaffold to explore in this context: a lathyrane diterpene. Although the full spectrum of activity of this novel class of activators on different PKC isozymes and leukemic cells remains to be investigated, our results with euphohelioscopin A hint that this natural product could have properties distinguishing it from currently known PKC activators.

Importantly, while PMA and related phorbol esters recapitulated the phenotype of euphohelioscopin A on CD34+ or myeloid leukemia cells, this was not the case for other PKC activators such as bryostatin-1 (Kraft et al., 1986), ADMB or ingenols (data not shown). This, together with the observation that euphohelioscopin A activates PKC-βII while also inducing myeloid differentiation (an activity shown to be mediated by different PKC isozymes but not PKC-βII (Mischak et al., 1993)), suggests that, like phorbol esters, euphohelioscopin A might be an activator of both conventional and novel PKCs. However, euphohelioscopin A also bears significant structural differences relative to PMA, including a less constrained structure than daphnane, ingenane and tigliane diterpenes (which comprise most known PKC-activating natural products isolated from Euphorbiaceae). Secondly, euphohelioscopin A lacks several functional groups that are thought to be important for the PKC binding and translocation induced by phorbol esters, such as an ester in position 13, the hydroxyl groups in positions 4, 9 and 20, and the carbonyl in position 3 (Blumberg, 1988; Nacro et al., 2000). Interestingly, kinetic differences in PKC activation were also observed between PMA and euphohelioscopin A: in addition to a 1000-fold lower potency, the latter showed a much slower rate of PKC activation (Figure 4B). This is particularly significant since lower rates of activation have been associated with differential phenotypic outcomes, including decreased PKC inactivation and subsequent degradation, a more sustained signal, and lower tumor promotion (Hampson et al., 2010).

These particularities highlight euphohelioscopin A and related lathyrane diterpenes as a family of natural products worthy of further investigation in the efforts toward safe and efficient pharmacological induction of myeloid differentiation.

SIGNIFICANCE

Through an unbiased cell-based screen with primary human CD34+ cells, we identified a novel activity of the plant natural product euphohelioscopin A. Euphohelioscopin A is able to induce myeloid differentiation in human hematopoietic stem and progenitor cells and in two myeloid leukemia cell lines. These findings suggest a potential utility of this scaffold for pharmacological intervention to alleviate a shortage or dysregulation of myeloid cells in the clinical setting. We were also able to determine the mechanism of this bioactivity by showing that euphohelioscopin A is an activator of protein kinase C; to our knowledge, this is the first evidence of a lathyrane diterpene activating PKC, and may shed new insights into the way ligands bind and activate this family of proteins.

EXPERIMENTAL PROCEDURES

CD34+ cell culture

All experiments were performed in HSC expansion media (StemSpan SFEM, StemCell Technologies) supplemented with 1x antibiotics and the following recombinant human cytokines: thrombopoietin, IL6, Flt3 ligand, and stem cell factor [100 ng/mL, R & D Systems]) unless otherwise indicated. Human mPB CD34+ cells were purified from fresh human leukophoresed mobilized peripheral blood (AllCells) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturer’s protocols. CD34+ cells were resuspended in HSC expansion medium (5×104 cells/mL) before being aliquoted in 384 well plates (Greiner Bio-One). Compounds were added immediately after plating. Cells were cultured at 37°C in 5% CO2.

Flow Cytometry

Cells were stained in staining medium (HBSS supplemented with FBS [2%] and EDTA [2 mM]) at 4°C for 1 h with PerCP anti-human CD34 (BD Bioscience), PECy7 anti-CD45ra (eBiosciences) and PE anti-CD11b (BD Bioscience), then washed with staining medium and analyzed. Multicolor analysis for cell phenotyping was performed on a LSR II flow cytometer (Becton Dickinson).

Colony forming assays

The progeny of 80 CD34+ cells after 7 days in culture were seeded in serum-free methylcellulose containing SCF, interleukin 6, erythropoietin, interleukin 3, granulocyte and granulocyte-macrophage colony stimulating factor (MethoCult SFH4436, StemCell Technologies), supplemented with 1x antibiotics, thrombopoietin and Flt3 ligand. Colonies were scored on day 14 with an inverted microscope at 40x magnification. Numbers reported represent the average of the number of colonies scored from three dishes.

Western Blots

2×106 HEK293T cells were serum starved overnight, then treated with Euphohelioscopin A, PMA or vehicle. After 10 minutes of compound treatment, cells were washed with cold PBS, then lysed with 100µL of RIPA buffer supplemented with a protease inhibitor cocktail (Sigma). Following incubation on ice for 20 min, the lysed cells were passed through a 26 gauge syringe needle and centrifuged (15,000g, 20 min at 4°C). Cell lysates were denatured by boiling in SDS sample buffer (Invitrogen) containing 5% β-mercaptoethanol. Proteins were electrophoresed, transferred onto a PVDF membrane and probed with anti-ERK1/2, anti-Phospho-ERK1/2, anti-MARCKS, anti-Phospho-MARCKS (all from Cell Signaling Technology), as well as anti-tubulin (Sigma) antibodies at suggested concentrations in Tris buffered saline containing 0.1% tween 20 and 5% nonfat dry milk. Blots were then incubated with HRP conjugated secondary antibodies and detected with a chemiluminescent substrate (Thermo Scientific).

Materials

Phorbol 12-myristate 13-acetate (PMA) and Gö 6983 were purchased from Calbiochem. Euphohelioscopin A was obtained from the Shanghai Institute of Materia Medica, Shanghai, China.

Cell culture

HEK293T and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (Cellgro) containing 10% fetal bovine serum (Gibco), at 37 °C, in 5% CO2. THP-1 and HL-60 cells were maintained in RPMI1640 (Cellgro) containing 10 % fetal bovine serum, at 37 °C, in 5% CO2.

Plasmid Constructs

The construction of CKAR and of C-terminally tagged rat PKC βII-RFP has been previously described(Gould et al., 2009; Violin et al., 2003).

Cell transfection

Cells were plated onto Lab-Tek chambered #1.0 borosilicate coverglass (Nunc) prior to transfection. Transient transfection of CKAR and rat PKC βII-RFP DNA was carried out using jetPRIME (Polyplus-transfection). Cells were imaged approximately 24 h following transfection.

FRET Imaging and Analysis

HeLa cells were rinsed with and imaged at room temperature in Hanks’ balanced salt solution (Cellgro) supplemented with 1 mM CaCl2. CFP, YFP, FRET, and RFP images were acquired and analyzed as described previously (Gallegos et al., 2006). RFP emission was monitored during the imaging experiments as a control for PKC expression levels. Base-line images were acquired for 3 or more minutes before ligand addition. PKC inhibitor Gö6983 was added 35 min after ligand addition. Individual data traces were normalized to 1 by dividing by the average base-line FRET ratio, and data from at least three different imaging dishes were referenced around the ligand addition time point. The normalized average FRET ratio is the average of these corrected values ± S.E.

Supplementary Material

Supplemental figure

ACKNOWLEDGEMENTS

We would like to thank Prof. Guo form the Shanghai Institute of Materia Medica for the supply of euphohelioscopin A. We thank Dr. Shoutian Zhu and Dr. Luke Lairson for helpful discussions and manuscript preparation. This work was supported by The Skaggs Institute for Chemical Biology (PGS) and NIH GM 43154 (ACN). CEA was supported by the National Science Foundation Graduate Research Fellowship under grant No. DGE1144086 and in part by the University of California at San Diego Graduate Training Program in Cellular and Molecular Pharmacology through NIGMS, National Institutes of Health Institutional Training Grant T32 GM007752.

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Supplementary Materials

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