Abstract
Although ingenol 3,20-dibenzoate (IDB) is known as a selective novel protein kinase C (PKC) agonist, its biologic actions and underlying mechanisms remain incompletely understood. In this study, we identified IDB as a proliferative agent for an erythropoietin (EPO)-dependent cell line, UT-7/EPO, through the screening of a natural compound library. To clarify the underlying mechanism of IDB’s EPO-like activities, we thoroughly analyzed the mutual relation between EPO and IDB in terms of in vitro and in vivo activities, signaling molecules, and a cellular receptor. IDB substantially induced the proliferation of UT-7/EPO cells, but not as much as EPO. IDB also lessened the anemia induced by 5-fluorouracil in an in vivo mouse model. Interestingly, IDB showed a synergistic effect on EPO at low concentration, but an antagonistic effect at higher concentration. Physical interaction and activation of PKCs by IDB- and EPO-competitive binding of IDB to EPO receptor (EPOR) explain these synergistic and antagonistic activities, respectively. Importantly, we addressed IDB’s mechanism of action by demonstrating the direct binding of IDB to PKCs, and by identifying EPOR as a novel molecular target of IDB. Based on these dual targeting properties, IDB holds promise as a new small molecule modulator of EPO-related pathologic conditions.
Introduction
The ingenol (IGN) diterpenoid derivatives from natural products have been reported to possess important biomedical activities, including anticancer (Challacombe et al., 2006; Jørgensen et al., 2013), antiviral (Hong et al., 2011), and thrombopoietic (Racke et al., 2012). In particular, IGN mebutate (Picato, PEP005, IGN 3-angelate) was recently approved by the U.S. Food and Drug Administration for actinic keratosis and was effective against human melanoma (Challacombe et al., 2006) and basal cell carcinomas (Siller et al., 2010). IGN protects human T cells from HIV infection (Hong et al., 2011), and IGN triacetate inhibited the replication of HIV strain (Fujiwara et al., 1996). IGN 3,20-dibenzoate (IDB) induces megakaryocytic differentiation (Racke et al., 2001, 2012). Nonetheless, the underlying mechanisms of the pleiotropic actions of IGN derivatives are not well understood.
IGN derivatives mimic endogenous diacylglycerol by activation and translocation of protein kinase C (PKC) that is responsible for a number of cellular signal transduction pathways and functions (Newton, 1995). PKC isozymes are classified into three groups: conventional PKC, which includes α, β, and γ isoforms; novel PKC (nPKC), which includes δ, ε, η, θ, and μ isoforms; and atypical PKC, which includes ζ and λ isoforms (Newton, 1995). IGN binds and activates partially purified PKC (Hasler et al., 1992), and IGN mebutate binds conventional PKC and nPKC (Kedei et al., 2004). However, the interactions between IGN derivatives and PKCs were only analyzed by the indirect method of [3H]phorbol 12,13-dibutyrate binding (Hasler et al., 1992; Kedei et al., 2004) or the docking simulation model (Grue-Sørensen et al., 2014). IDB can selectively activate nPKC (Asada et al., 1998), but direct binding kinetics have never been measured. Instead of PKC as a molecular target, evidence for the existence of cell membrane receptors for phorbol or IGN esters was suggested over 30 years ago (Driedger and Blumberg, 1980; Dunphy et al., 1980; Shoyab and Todaro, 1980; Sando et al., 1981), but they have not been identified to date.
Human erythropoietin (EPO) is the main cytokine that stimulates proliferation and differentiation of erythroid progenitor cells. EPO receptor (EPOR) mediates the activities of EPO. EPO binding to EPOR induces a conformational change, which results in phosphorylation of Janus tyrosine kinase-2 (JAK-2), EPOR itself, and other signal-transduction proteins, including the signal transducer and activator of transcription-5 (Constantinescu et al., 2001). PKC appears to play an important role in EPOR signaling, especially for the activation of RAF-1 and extracellular signal-regulated kinase (ERK)1/2 (von Lindern et al., 2000; Szenajch et al., 2010).
Recombinant EPO protein and its modified forms have been successfully used in the clinic to treat anemia. Because of some of EPO’s limitations, including the existence of EPO-resistant anemia, pure red cell aplasia, tumor-promoting effects, high cost, and invasive route of administration, researchers are trying to develop alternative EPOR-activating molecules (Kim et al., 2013). Small molecules and peptide EPO mimetics that activate EPOR have been reported (Qureshi et al., 1999; Goldberg et al., 2002). However, until now, small molecule mimetics were not effective enough to be used as a therapeutic agent, and the development of peptide mimetics is underway (Kessler et al., 2012).
UT-7/EPO is a human erythroleukemia cell line that is absolutely dependent on EPO for long-term maintenance (Komatsu et al., 1993). We and other researchers have used the UT-7/EPO cell line for the EPO bioassay, EPO signaling study, and EPO-mimetic drug test (Fan et al., 2006; Liu et al., 2007; Shin et al., 2010, 2012; Kessler et al., 2012). Little information is available regarding nonpeptide small molecules with UT-7/EPO proliferative activities.
Although several herbal extracts promote erythropoiesis, most of their direct molecular targets are unidentified due to the presence of a large number of active compounds in the extracts. Using synthetic chemical libraries, researchers have tried to discover the single active compounds, but have been unsuccessful up to this point (Qureshi et al., 1999; Goldberg et al., 2002). Considering the diverse and complex conformations of ingredients of natural products, we believe that identification of EPO-mimetic small molecules from the natural world is promising.
In the present study, we discover IDB possesses EPO-modulatory properties and investigate its underlying mechanism of actions by identifying direct molecular targets.
Materials and Methods
EPO Bioassay System for Screening the Natural Product Library.
A commercial library with 502 purified natural products of known structure (BML-2865; Enzo Life Sciences, Farmingdale, NY) was screened using UT-7/EPO cells (Shin et al., 2010). UT-7/EPO cells were maintained in α-minimal essential medium (Hyclone, Logan, UT), supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY) and 1 IU/ml erythropoietin (EPO; LG Life Science, Seoul, Korea) in a humidified CO2 incubator. UT-7/EPO cells were washed three times with α-minimal essential medium, and 104 cells in EPO-free culture medium were added in triplicate to wells and starved for 1 day. After starvation, 0.2 IU/ml EPO or 20 μg/ml herbal compound was added to the cells and incubated for 3 days at 37°C in a CO2 incubator. After incubation, EZ-Cytox cell viability water-soluble tetrazolium salt assay kits (Daeil Laboratory Service, Seoul, Korea) were used to measure cell proliferation. Fifteen microliters of kit reagent was added to each well and incubated for 1–6 hours in a CO2 incubator. After incubation, optical density was measured at an absorbance of 450 nm (reference wavelength: 690 nm).
Bioassay for Analyzing the Dose-Dependent Activities of IDB and EPO.
A proliferative response of UT-7/EPO cells to EPO, IDB (Santa Cruz Biotechnology, Santa Cruz, CA; Enzo Life Sciences), or IGN (Santa Cruz Biotechnology) was measured. EPO (4-fold dilutions from 5 IU/ml), IDB (4-fold dilutions from 5 μg/ml), or IGN (4-fold dilutions from 5 μg/ml) was added to the cells and incubated for 3 days. A viability assay was performed, as described above.
Analysis of the Activity of IDB or EPO on Human Bone Marrow CD34+ Progenitor Cells.
Human bone marrow CD34+ progenitor cells (1 × 104 cells) (Lonza, Walkersville, MD) were incubated with EPO (4 IU/ml), IDB (1 μg/ml), or IGN (1 μg/ml) in StemSpan SFEM supplemented with cytokine cocktail CC100 (StemCell Technologies, Vancouver, Canada) in a 12-well tissue culture dish. After incubation at 37°C, 5% CO2, for 10 days, viability assays were performed, as described above.
In Vivo Model of 5-Fluorouracil–Induced Anemia for Analyzing the Activities of IDB and EPO.
Male C57BL/6 mice (8–12 weeks old) were used (five mice per treatment group). On day 0, anemia was induced in mice via a single intraperitoneal injection of 3 mg 5-fluorouracil (5-FU; Sigma-Aldrich, St. Louis, MO). On days 3, 7, and 11, mice were administered EPO (100 IU), IDB (20 μg), or vehicle in 0.2 ml saline. Blood samples were collected via the retro-orbital plexus at the indicated time points (days 0, 4, 8, 12, 16, and 20) using a heparinized capillary. Hematologic parameters were measured using a Procyte DX hematology analyzer (IDEXX Laboratories, Westbrook, ME). Two independent experiments were performed. Animal studies were approved by the Department of Laboratory Animal, Institutional Animal Care and Use Committee at Sungsim campus of Catholic University of Korea (Bucheon, Korea).
Bioassay for Analyzing the Activity of an EPO and IDB Mixture.
UT-7/EPO cell proliferative activity of an EPO and IDB mixture was measured. IGN (0.078 μg/ml or 1.25 μg/ml), IDB (0.078 μg/ml or 1.25 μg/ml), EPO (0.078 IU/ml or 1.25 IU/ml), or mixture of EPO (0.078 IU/ml) and IDB (0.078 μg/ml) or EPO (1.25 IU/ml) and IDB (1.25 μg/ml) were applied to UT-7/EPO cell bioassay, as described above.
Erythroid Colony Assay.
MethoCult assay media for human colony-forming cell assay were prepared by adding 50 ng/ml human stem cell factor, 20 ng/ml human granulocyte-macrophage colony-stimulating factor, 20 ng/ml human interleukin-3, 20 ng/ml human interleukin-6, and 20 ng/ml human granulocyte colony-stimulating factor (PeproTech, Rocky Hill, NJ) into MethoCult media (Stemcell Technologies). Human bone marrow CD34+ progenitor cells (1 × 104 cells) (Lonza) were incubated with 0.3 ml EPO (30 IU/ml), IDB (0.03 or 1 μg/ml), or a mixture in MethoCult assay media in 35-mm dishes. After incubation at 37°C in 5% CO2 for 12∼14 days, erythroid (BFU-E) colonies were counted using inverted microscope (Zeiss, Oberkochen, Germany).
Western Blot for Analyzing the PKC Activation Signaling by IDB.
EPO- or IDB-induced translocation of PKC-α, PKC-δ, or PKC-ε to cellular membrane was analyzed by Western blot (WB). EPO-starved UT-7/EPO cells (2 × 106 cells) were prepared on 100-mm plates and treated with EPO (3 IU/ml), IDB (3 μM), IGN (3 μM), or vehicle for 24 hours. Membrane proteins and cytosolic proteins were prepared with MEM-per plus membrane protein extraction kit (Thermo Scientific, Rockford, IL), according to the manufacturer’s instructions. From each sample, 30 μg protein was electrophoresed on a SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The protein-containing membrane was blocked using superblock T20 blocking buffer (Thermo Scientific) and then probed with anti–PKC-α antibodies (Santa Cruz Biotechnology), anti–PKC-δ (Cell Signaling, Beverly, MA), or anti–PKC-ε (Cell Signaling). Primary antibodies were diluted 1:500 or 1:1000 in Tris-buffered saline with 5% skim milk (BD Biosciences, San Jose, CA) and 0.1% Tween 20 (Sigma-Aldrich). Anti-mouse IgG horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) and Super Signal West Pico Luminal/Enhancer solution (Pierce, Rockford, IL) were used for chemiluminescence. All bands were detected using chemidoc XRS+ system (Bio-Rad, Richmond, CA).
Bioassay for Analyzing the Inhibition of EPO or IBD Activity by PKC Inhibitors.
Inhibition of UT-7/EPO cell proliferative activity of EPO or IDB by PKC inhibitors was measured. Gö6976 [5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile] (Calbiochem, Somerville, NJ) or bisindolylmaleimides (BIMs; Merck, Darmstadt, Germany) were serially diluted 2-fold from 10 μM stock and added to cells. After a 30-minute incubation in a CO2 incubator, EPO (0.25 IU/ml) or IDB (0.8 μg/ml) was applied to the UT-7/EPO cell bioassay, as described above.
Surface Plasmon Resonance for Analyzing the IDB Interaction with PKC-α and PKC-ε.
To monitor the binding kinetics between IDB and human PKC-α (Abcam, Cambridge, MA) or PKC-ε (Abcam), surface plasmon resonance (SPR) analysis was performed using Biacore T200 model (GE Healthcare, Princeton, NJ) at 25°C with buffer HBS-ET+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20) containing 5% dimethylsulfoxide. The pH scouting to immobilize PKC isoforms was performed in 10 mM acetate buffer at pH 4.0, 4.5, 5.0, and 5.5. PKC-α was immobilized on a CM5 sensor chip up to 5012 response units (RU), and PKC-ε was up to 3356 RU with the standard amine-coupling method at pH 4.5. IDB was injected into the PKC isoform-immobilized flow cell at concentrations of 6.25, 3.13, 1.56, 0.78, and 0.39 μM with a flow rate of 20 μl/min for 45 seconds, and allowed to dissociated for 180 seconds. T-200 BIAevaluation software was used to subtract references and determine a steady state affinity constant (KD). Between sample series, a solvent correction cycle was run to adjust for referencing errors due to refractive index mismatches between running buffer and samples.
Bioassay for Analyzing the Inhibition of EPO Activity by Increasing Amount of IDB.
The inhibition of EPO-induced UT-7/EPO cell proliferation by increasing concentrations of IDB was measured. EPO (1 IU/ml) was coincubated with IDB or IGN (4-fold serial dilutions from 5 μg/ml stock) and applied to the UT-7/EPO cell bioassay, as described above.
Bioassay for Analyzing the Inhibition of IDB Activity by Soluble EPOR.
The inhibition of EPO- and IDB-induced cell proliferation by soluble EPOR (sEPOR) was monitored. UT-7/EPO cells were incubated with IDB (0.8 μg/ml) or EPO (0.25 IU/ml) in the presence of sEPOR (2-fold serial dilutions from 2.5 μg/ml stock) and applied to the UT-7/EPO cell bioassay, as described above.
Enzyme-Linked Immunosorbent Assay for Analyzing the Inhibition of EPO Binding to sEPOR with IDB.
sEPOR was diluted to 1 μg/ml in phosphate-buffered saline (PBS) and coated on a 96-well Maxisorp Nunc Immunoplate (Nunc, Roskilde, Denmark) at 4°C overnight. After washing with 0.05% Tween 20 in PBS using an automated washer (Bio-Rad), the plates were blocked with PBS containing 1% bovine serum albumin (Sigma-Aldrich) for 1 hour at room temperature. After washing, 5 μg/ml EPO in PBSAT [1% bovine serum albumin and 0.05% Tween 20 in PBS] or mixtures of 5 μg/ml EPO plus IDB (25, 50, 100, or 200 μg/ml) were added to each well and incubated for 1 hour at room temperature. After washing, anti-EPO antibodies (Abcam) diluted in PBSAT (1:200) were added to each well and incubated for 1 hour. After washing, rabbit anti-mouse horseradish peroxidase antibodies (Santa Cruz Biotechnology) diluted in PBSAT (1:2500) were added to each well and incubated for 1 hour. After washing, 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt solution (Roche, Mannheim, Germany) was added to each well, and optical density was measured at an absorbance of 405 nm.
WB for Analyzing the Inhibition of IDB or EPO Signaling by sEPOR.
Inhibition of IDB- or EPO-induced ERK1/2 phosphorylation by sEPOR coincubation was analyzed by WB. Starved UT-7/EPO cells (2 × 106 cells) were treated with EPO (0.25 IU/ml), IDB (0.8 μg/ml), or vehicle in the presence or absence of sEPOR (5 μg/ml) for 5 minutes. Total proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Fisher Scientific, Pittsburgh, PA) and applied to the WB assay, as described above. Anti-ERK1/2 (Thermo Scientific) and phospho-ERK1/2 (Thermo Scientific) were used for primary antibodies.
WB for Analyzing IDB-Induced Phosphorylation of EPOR and ERK1/2 in the Presence or Absence of EPO.
Starved UT-7/EPO cells (2 × 106 cells) were treated with various concentrations of IDB (1.6, 0.8, 0.4, 0.2, and 0.1 μg/ml) with or without EPO (0.25 IU/ml) for 5 minutes. Total proteins were extracted using RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Fisher) and applied to the WB assay, as described above. Anti-EPOR (Santa Cruz Biotechnology), phospho-EPOR (Santa Cruz Biotechnology), ERK1/2, and phospho-ERK1/2 were used for primary antibodies.
WB for Analyzing the IDB-Induced Phosphorylation of JAK-2.
Starved UT-7/EPO cells (2 × 106 cells) were treated with EPO (1, 0.25, or 0.0625 IU/ml), IDB (1.6, 0.4, or 0.1 μg/ml), or IGN (1.6, 0.4, or 0.1 μg/ml) for 5 minutes. Total proteins were extracted using RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Fisher) and applied to the WB assay, as described above. Anti–JAK-2 (Cell Signaling) and phospho–JAK-2 (Cell Signaling) were used for primary antibodies.
SPR for Analyzing the IDB Binding to sEPOR.
To analyze the interaction between IDB and human sEPOR, the pH scouting for immobilization of recombinant sEPOR was performed in 10 mM acetate buffer at pH 4.0, 4.5, 5.0, and 5.5. sEPOR was immobilized on a CM5 sensor chip up to 1612 RU with a standard amine-coupling method at pH 5.0. IDB was injected into the sEPOR-immobilized flow cell at concentrations of 6.25, 3.13, 1.56, 0.78, and 0.39 μM with a flow rate of 20 μl/min for 45 seconds and allowed to dissociate for 180 seconds.
Data Analysis.
Results are expressed or plotted as the mean ± S.D. or S.E.M., and data were compared for statistical significance with Student’s t test or one-way analysis of variance, and *P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant.
Results
EPO-Mimetic Properties of IDB in Cell and Mouse Models.
Through the screening of a natural compound library, IGN IDB was identified as an active compound that induced proliferation of the EPO-dependent cell line, UT-7/EPO (Fig. 1A). IDB (Fig. 1B) and IGN (Fig. 1C) are diterpenoids related to phorbol, and IDB is a known PKC-ε activator and IGN is a weak activator of PKC-ε and PKC-δ. Despite the same core structure between IDB and IGN, IGN did not induce UT-7/EPO proliferation (Fig. 1D). IDB dose-dependently promoted UT-7/EPO cell proliferation with an EC50 of 0.27 μg/ml (485 nM) (Fig. 1D). The maximum effect of IDB was about 70% that of EPO. Additionally, we assessed the IDB-driven expansion of human bone marrow CD34+ progenitor cells. Like EPO, IDB also positively affects the proliferation of primary CD34+ cells (Fig. 1E).
IGN IDB stimulates the proliferation of the UT-7/EPO cell line with absolute dependence on EPO as well as human bone marrow CD34+ progenitor cells. (A) Compound library from natural products was screened using UT-7/EPO cells to identify a compound with proliferative activity. After EPO starvation for 24 hours, UT-7/EPO cells were incubated with EPO (0.2 IU/ml) or one of the library compounds (20 μg/ml), and cell viability was measured by water-soluble tetrazolium salt assay. IDB was identified as a hit of about 400 compounds, and representative data are shown. (B) Chemical structure of IDB. (C) Chemical structure of IGN used as a control. (D) Dose-dependent enhancement of UT-7/EPO proliferation by IDB. EPO (5 IU/ml), IDB (5 μg/ml), or IGN (5 μg/ml) was serially diluted and applied to UT-7/EPO cell proliferation assay. Mean optical density (OD) values of triplicate samples ± S.D. are shown. (E) Stimulation of proliferation of human bone marrow CD34+ progenitor cells by IDB. Human bone marrow CD34+ progenitor cells were incubated with EPO (4 IU/ml), IDB (1 μg/ml), or IGN (1 μg/ml) in StemSpan SFEM supplemented with cytokine cocktail CC100. After a 10-day incubation, viability assays were performed. Mean optical density values of triplicate samples ± S.E.M. are shown. *P < 0.05, ***P < 0.001, with respect to the vehicle-treated group.
Given that IDB was shown to have EPO-like capacity in promoting the proliferation of a solely EPO-dependent cell line, its performance was assessed in a 5-FU–induced anemia model (Fig. 2). Overall, severity of 5-FU–induced anemia was lessened by IDB treatment on days 12 and 20 (Fig. 2, A, C, and E), which was shown by the significant elevation of red blood cells, hematocrit, and hemoglobin levels compared with vehicle-treated group (Fig. 2, B, D, and F). IDB also elevated reticulocytes on day 16 (Fig. 2, G and H).
In vivo activity of IDB. Anemia was induced by an intraperitoneal injection of 5-FU (3 mg/mouse) into C57BL/6 mice on day 0. EPO (100 IU/mouse), IDB (20 μg/mouse), or vehicle was administered on days 3, 7, and 11. Hematologic parameters, including red blood cells (RBC; 106 cells/μl) (A and B), hematocrit (HCT; %) (C and D), hemoglobin (Hb; g/dl) (E and F), reticulocytes (RET; 103 cells/μl) (G and H), and platelets (PLT; 103 cells/μl) (I and J), were measured using a Procyte DX hematology analyzer. Representative data from two independent experiments are shown as the mean ± S.E.M. from five mice for vehicle-, EPO-, and IDB-treated groups. *P < 0.05, **P < 0.01, compared with values from vehicle-treated mice.
Both Synergistic and Antagonistic Effects of IDB against EPO.
To check whether the activities of EPO and IDB are either cooperative or competitive, a bioassay was performed with the subsaturated concentration mixture of 0.08 IU/ml EPO and 0.08 μg/ml IDB (Fig. 3A) or with the saturated one of 1.25 IU/ml EPO and 1.25 μg/ml IDB (Fig. 4A). Although IDB and EPO synergistically stimulated UT-7/EPO cell proliferation at a subsaturated concentration (Fig. 3A), IDB suppressed EPO activity in cells similarly to those treated with IDB alone at saturated concentrations (Fig. 4A). We demonstrated this outcome in more detail with mixtures of concentration-fixed EPO (1 IU/ml) and serially diluted IDB (Fig. 4B). EPO activity was completely downregulated by IDB in a dose-dependent manner, but IDB’s own activity was maintained (Fig. 4B). Similarly, synergistic (Fig. 3B) or antagonistic activity (Fig. 4C) between EPO and IDB was examined in an ex vivo assay. Although 0.003 μg/ml IDB alone was unable to stimulate the differentiation of human bone marrow CD34+ cells into BFU-E colonies, it significantly synergized EPO activity (Fig. 3B). In contrast, 0.1 μg/ml IDB suppressed EPO-induced BFU-E colony formation (Fig. 4C). To understand the differential actions of IDB on EPO, we began to investigate the underlying mechanisms.
EPO synergistic activity of IDB is attributable to the direct targeting of PKC. (A) Promotion of EPO activity with IDB in a bioassay. IGN (0.08 μg/ml), IDB (0.08 μg/ml), EPO (0.08 IU/ml), or a mixture of EPO (0.08 IU/ml) and IDB (0.08 μg/ml) was applied to the UT-7/EPO cell proliferation assay. Mean optical density (OD) values of triplicate samples ± S.D. are shown. **P < 0.01, ***P < 0.001 in comparison with EPO-alone group. (B) Stimulation of the differentiation of human CD34+ bone marrow cells into BFU-E colonies by EPO or EPO plus IDB. CD34+ cells were incubated for 12∼14 days in MethoCult media in the presence of IDB (0.003 μg/ml), EPO (3 IU/ml), or IDB (0.003 μg/ml) plus EPO (3 IU/ml). BFU-E colonies were counted using an inverted microscope (left). Representative images were taken at 100× magnification (right). (C) Translocations of PKCs to the cell membrane (Memb) were analyzed by WB after treatment with EPO (3 IU/ml), IDB (3 μM), or IGN (3 μM) for 24 hours. WB bands were quantified by densitometric analysis. (D and E) Inhibition of cell proliferative activity of EPO and IDB by PKC inhibitors. Gö6976 (D) or BIM (E) was serially diluted 2-fold from 10 μM stock and added to the cells. After a 30-minute incubation in a CO2 incubator, EPO (0.25 IU/ml) or IDB (0.8 μg/ml) was added to each well and incubated for another 72 hours. Cell proliferation was measured by water-soluble tetrazolium salt assay. (F–I) SPR analysis was performed using a BIAcore instrument. PKC-α or PKC-ε was immobilized on a CM5 sensor chip with the standard amine-coupling method, and IDB or IGN was injected to the PKC-coated flow cell at indicated concentrations. T-200 BIAevaluation software was used to subtract references and determine the affinity constant (KD).
EPO inhibitory activity of IDB is attributable to the competitive binding of IDB and EPO toward EPOR. (A) Inhibition of EPO activity with a high concentration of IDB in a bioassay. IGN (1.25 μg/ml), IDB (1.25 μg/ml), EPO (1.25 IU/ml), or a mixture of EPO (1.25 IU/ml) and IDB (1.25 μg/ml) was applied to the UT-7/EPO cell proliferation assay. Mean optical density (OD) values of triplicate samples ± S.D. are shown. **P < 0.01, ***P < 0.001 in comparison with EPO-alone group. (B) Inhibition of activity using a fixed concentration of EPO with increasing concentrations of IDB or IGN. A quantity amounting to 1 IU/ml EPO was coincubated with increasing amounts of IDB or IGN up to 5 μg/ml and applied to the UT-7/EPO cell proliferation assay. For comparison, serial dilutions of IDB or IGN were applied to cells in the absence of EPO. Mean optical density values of triplicate samples ± S.D. are shown. (C) Suppression of EPO-induced differentiation in human CD34+ bone marrow cells into BFU-E colonies by a high concentration of IDB. CD34+ cells were incubated for 12∼14 days in MethoCult media in the presence of IDB (0.1 μg/ml), EPO (3 IU/ml), or IDB (0.1 μg/ml) plus EPO (3 U/ml). (D) Inhibition of cell proliferative activity by IDB or EPO with sEPOR. UT-7/EPO cells were incubated with IDB (0.8 μg/ml) or EPO (0.25 IU/ml) in the presence of sEPOR (2-fold dilutions from 2.5 μg/ml), and the water-soluble tetrazolium salt assay was performed. Mean optical density values of triplicate samples ± S.D. are shown. (E) Inhibition of EPO binding to plate-coated sEPOR by IDB. Enzyme-linked immunosorbent assay plate was coated with sEPOR (1 μg/ml) and added by EPO (5 μg/ml) in the presence of serially diluted IDB (200, 100, 50, 25 μg/ml). After washing, bound EPO was detected by anti-EPO primary antibodies. Mean optical density values of triplicate samples ± S.D. are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistical significance in comparison with cells treated with EPO alone. (F) Inhibition of IDB- or EPO-induced ERK1/2 phosphorylation by coincubation with sEPOR. ERK1/2 signaling in UT-7/EPO cells stimulated by IDB (0.8 μg/ml) or EPO (0.25 IU/ml) in the presence of sEPOR (5 μg/ml) for 5 minutes was analyzed by WB. WB bands were quantified by densitometric analysis, and the data are presented as the mean ± S.D. of three experiments. Data were analyzed by Student t test (*P < 0.05; **P < 0.01; ***P < 0.001). (G) Dose-dependent inhibition of EPO-induced EPOR phosphorylation by IDB. Effect of IDB (1.6, 0.8, 0.4, 0.2, or 0.1 μg/ml) coincubation in the EPO (0.25 IU/ml)-induced phosphorylation of EPOR and ERK1/2 was analyzed by WB. WB bands were quantified by densitometric analysis, and the data are presented as the mean ± S.D. of three experiments. Data were analyzed by one-way analysis of variance with Dunnett post-test (*P < 0.05; **P < 0.01; ***P < 0.001). (H) Inefficiency of JAK-2 phosphorylation by IDB. WB analysis of JAK-2 phosphorylation by EPO (1, 0.25, or 0.0625 IU/ml), IDB (1.6, 0.4, or 0.1 μg/ml), or IGN (1.6, 0.4, or 0.1 μg/ml) was performed. WB bands were quantified by densitometric analysis, and the data are presented as the mean ± S.D. of three experiments. Data were analyzed by one-way analysis of variance with Dunnett's post-test (*P < 0.05; **P < 0.01; ***P < 0.001). (I and J) SPR analysis was performed using BIAcore. sEPOR was immobilized on a CM5 sensor chip with a standard amine-coupling method, and IDB or IGN was injected into the sEPOR-coated flow cell at indicated concentrations. T-200 BIAevaluation software was used to subtract references and determine the affinity constant (KD).
Synergistic Effects of IDB at Subsaturated Concentration with EPO via Direct Binding to PKCs.
At first, the effects of IDB on the activation and membrane translocation of PKC isoforms were studied in UT-7/EPO cells (Fig. 3C). PKC-α, PKC-δ, and PKC-ε were translocated to the membrane fraction in IDB-treated cells, and EPO slightly activated PKC-δ and PKC-ε. No PKCs tested by us were affected by IGN treatment. Next, the effect of a classic PKC inhibitor on the IDB- or EPO-mediated UT-7/EPO cell proliferation was assessed (Fig. 3, D and E). Whereas EPO-induced cell proliferation was almost completely blocked by Gö6976, a PKC-α and PKC-β inhibitor, in a dose-dependent manner, IDB-induced proliferation was partially blocked (Fig. 3D). BIM, a general PKC inhibitor, was added to EPO- or IDB-treated cells (Fig. 3E). Although both activities of EPO and IDB were almost completely inhibited by BIM treatment in a dose-dependent manner, IDB activity was much more sensitive (Fig. 3E). Further evidence of IDB’s action on PKCs was provided by SPR analysis by BIAcore (Fig. 3, F–I). Purified PKC-α or PKC-ε was covalently cross-linked to the dextran matrix of a CM5 sensor chip, and various concentrations of IDB were passed over this surface as well as the reference surface. Representative reference-subtracted overlaid sensorgrams are displayed. IDB bound directly to PKC-α (Fig. 3F) and PKC-ε (Fig. 3H) with similar binding affinities (KD), but IGN did not (Fig. 3, G and I).
IDB Competes and Interferes with EPO via Direct Binding to EPOR.
It was observed that a relatively high amount of IDB downregulated EPO activity nearly to the level of the group treated with IDB alone (Fig. 4, A and B). We postulated that most activities of EPO (1 IU/ml) plus IDB (high concentrations) seem to be attributed by remaining IDB that targets PKCs, but not by IDB that participates in the EPO blockade via direct competition for the EPOR.
To prove this hypothesis, we investigated the impact of soluble EPOR on IDB-related activity. IBD’s cell proliferation activity was partially hampered by sEPOR coincubation, whereas the activity of EPO was almost completely suppressed by sEPOR (Fig. 4D). Partial inhibition of IDB activity by sEPOR coincubation led to the idea that it may be possible for IDB to target at least two molecules, sEPOR and PKCs. To determine whether IDB competes with EPO in binding to sEPOR at a molecular level, enzyme-linked immunosorbent assay was used to analyze the interaction between EPO and sEPOR. Protein-protein interaction of EPO and sEPOR was substantially disrupted by the addition of IDB to the EPO sample in a dose-dependent manner (Fig. 4E). In addition, it was demonstrated that sEPOR was able to block EPO- and IDB-induced phosphorylation of ERK1/2, most likely through direct binding to EPO or IDB (Fig. 4F).
Because preceding data have shown that IDB possibly binds directly to EPOR, we tested whether IDB is able to stimulate the internal EPOR-related signaling. IDB stimulated the phosphorylation of ERK1/2, but not the phosphorylation of EPOR and JAK-2 (Fig. 4, G and H). By demonstrating the blockade of EPO-induced EPOR phosphorylation by IDB (Fig. 4G) and the specific binding kinetics between IDB and EPOR with a KD of 4.02 × 10−6 M (Fig. 4, I and J), we collectively concluded that IDB does bind to EPOR in a competitive fashion with EPO, but does not activate the EPOR-initiated signaling pathway.
Discussion
To find an EPO-mimetic agent from natural sources, an herbal compound library was screened using an EPO-dependent cell line, and it is from this work that IDB was discovered. To our knowledge, the data first showed the dual functions of IDB, EPO-supportive activity via PKC, and EPO-suppressive activity via the EPOR. It was previously reported that EPO-mimetic synthetic compounds from chemical collections exhibited much less potency than EPO (under 20% that of EPO) with no in vivo data (Qureshi et al., 1999; Goldberg et al., 2002), but we demonstrated the substantial in vitro efficacy of IDB (about 70% that of EPO) with an in vivo hematopoietic activity (Figs. 1 and 2).
Although very recent research regarding IGN derivatives shed a new light on the chemical synthesis (Jørgensen et al., 2013; McKerrall et al., 2014) and clinical application (Challacombe et al., 2006; Siller et al., 2010), their mode of action is still obscure. Moreover, the data about IDB are much more limited in terms of activities and their mechanisms. Unlike the known agonistic action of IDB on the nPKCs (Racke et al., 2001, 2012), IDB also seems to activate classic PKCs, which was demonstrated by the partial inhibition of IDB activity by a PKC-α inhibitor (Fig. 3D), complete inhibition by a broad PKC inhibitor (Fig. 3E), and the physical interactions of IDB with PKC-α and PKC-ε (Fig. 3, F and H). IDB possesses erythropoietic activities in vivo (Fig. 2), and this effect is likely attributable to the EPO-mimic activation of PKC (von Lindern et al., 2000). Usually, binding affinity of chemical PKC agonists was measured by the inhibition of [3H]phorbol 12,13-dibutyrate binding (Hasler et al., 1992; Kedei et al., 2004), but we first adopted SPR analysis to demonstrate the direct binding of IDB to PKC-α and PKC-ε (Fig. 3, F and H). IGN did not bind to PKC-α and PKC-ε and did not show any biologic activities.
Although the synergistic effect of IDB and EPO on UT-7/EPO proliferation appears to be implicated in the targeting of PKC and EPOR, respectively, EPO antagonistic effect (Fig. 4, A–C) of IDB could not be explained with PKC targeting alone. Considering the EPO mimicking, but competitive effects of IDB, we placed the EPOR on the list of novel target suspects. IDB does directly bind to EPOR (Fig. 4I), which could interfere with the EPO binding to EPOR (Fig. 4E) and a subsequent blockade of EPO signaling (Fig. 4G). In addition, sEPOR was able to bind to IDB, blocking its cellular proliferation (Fig. 4D) and downstream signaling activity (Fig. 4F). IDB is not regarded as an EPOR agonist, but as an antagonist, because it failed to stimulate JAK-2 and EPOR phosphorylation (Fig. 4, G and H). Although some synthetic small molecules as a multimeric form stimulated EPO-responder cells, direct evidence regarding the triggering of internal signaling was missing (Qureshi et al., 1999; Goldberg et al., 2002). This is the first report that IGN ester, a PKC agonist, could target and antagonize EPOR.
The dual role of IDB as an EPO-synergizing (Fig. 3) or EPO-suppressing molecule (Fig. 4) is due to the dual targeting of PKC and EPOR in a concentration-dependent manner by IDB and EPO. At limiting concentrations of IDB and EPO, IDB tends to strengthen the intracellular PKC/ERK signaling of EPO (Fig. 4G) by direct activation of PKC (Fig. 3, C, F, and H), rather than compete with EPO for EPOR binding due to the possible availability of EPO-unbound EPOR on the cell surface, resulting in enhanced stimulation of cell proliferation by the mixture of IDB and EPO (Fig. 3A). At saturating concentrations of IDB and EPO, IDB appears to inhibit the cell surface binding of EPO to EPOR by competitively binding to EPOR (Fig. 4, D, E, and I). IDB also appears to sustain the PKC/ERK activation signal (Fig. 4G) via excess IDB that does not participate in EPOR binding, but instead penetrates the membrane and binds to PKC, resulting in suppressed stimulation of cell proliferation by the mixture of IDB and EPO (Fig. 4, A and B).
The antagonistic activity of IDB against EPO provides a speculation that IDB may counteract the potential deleterious effects of EPO, such as the growth of some EPO-dependent tumors (Pérès et al., 2011; Wu et al., 2012), increased mortality rate of cancer patients treated with recombinant EPO for cancer-associated anemia (Henke et al., 2003; Leyland-Jones et al., 2003; Wright et al., 2007; Bennett et al., 2008), and high endogenous EPO levels related to certain diseases, such as myelodysplastic syndrome (Wallvik et al., 2002; Moyo et al., 2008). IDB and IDB derivatives induce apoptosis in Jurkat leukemia cells (Blanco-Molina et al., 2001), and IGN mebutate is effective against actinic keratosis, human melanoma (Challacombe et al., 2006), and basal cell carcinomas (Siller et al., 2010). Considering the intrinsic hematopoietic, anticancer, and EPOR antagonistic activities of IDB, researchers believe that utilizing IDB to treat cancer-related anemia is a promising approach.
In conclusion, we have identified a new biologic effect and IDB target. These findings could provide novel insights into the underlying mechanisms of biologically active IGN derivatives and could be the basis for the development of novel therapeutic strategies for EPO-related disorders.
Acknowledgments
The authors thank Professor Il-Hoan Oh for experimental advice and Dr. Sunny S. Choe for reading and correcting the manuscript.
Authorship Contributions
Participated in research design: Oh, S. K. Kim, S.-J. Kim, Kang, Heo.
Conducted experiments: Oh, Choi.
Contributed new reagents or analytic tools: Chin.
Performed data analysis: Oh, Kang, Heo.
Wrote or contributed to the writing of the manuscript: Oh, S. K. Kim, Heo.
Footnotes
- Received December 14, 2014.
- Accepted June 5, 2015.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education [2013R1A1A2009176]; the Bio and Medical Technology Development Program of the NRF funded by the Ministry of Science, Information and Communication Technology, and Future Planning [NRF-2013M3A9B5075839]; and Research Fund 2011 of the Catholic University of Korea.
Abbreviations
- BIM
- bisindolylmaleimide
- EPO
- erythropoietin
- EPOR
- EPO receptor
- ERK
- extracellular signal-regulated kinase
- 5-FU
- 5-fluorouracil
- Gö6976
- 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile
- IDB
- IGN 3,20-dibenzoate
- IGN
- ingenol
- JAK-2
- Janus tyrosine kinase-2
- nPKC
- novel PKC
- PBS
- phosphate-buffered saline
- PKC
- protein kinase C
- RIPA
- radioimmunoprecipitation assay
- RU
- response unit
- sEPOR
- soluble EPOR
- SPR
- surface plasmon resonance
- WB
- Western blot
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
References
In this issue
IDB Targets Both PKC and EPO Receptor
