Requirement for PLCg2 in IL-3 and GM-CSF-Stimulated MEK/ERK Phosphorylation in Murine and Human Hematopoietic Stem/ Progenitor Cells
Studies suggest an involvement of intracellular Ca2þ (Ca2þ) in mediating the cytokine signals that control hematopoiesis (Whetton et al., 1988a; Ren et al., 1994; Collison et al., 1998; Tong et al., 2004; Paredes-Gamero et al., 2008). Ca2þ is an important second messenger in many physiological processes such as proliferation, differentiation, and cell death (Berridge et al., 2003; Berridge, 2006, 2009). Tyrosine receptor- dependent Ca2þ release is mediated by phospholipase Cg (PLCg) activation that occurs in response to its recognition of phosphorylated tyrosine residues on the cytokine receptors of hematopoietic cells (Wilde and Watson, 2001). Activation of PLCg results in the hydrolysis of phosphatidylinositol 4,5- bisphosphate (PIP2), thus forming inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) as second messengers (Berridge and Irvine, 1984; Wahl et al., 1988). These molecules lead to Ca2þ release from intracellular stores and protein kinase C 2002; Kitsos et al., 2005; Berridge, 2006; Paredes-Gamero et al., 2006; Roderick and Cook, 2008).
Among Ca2þ sensitive proteins, the participation of PKC was first described in the differentiation process of hematopoietic precursors. High concentrations of phorbol 12-myristate 13-acetate (PMA) induced myelo/monocyte differentiation, whereas low concentrations induced eosinophil differentiation (Rossi et al., 1996). In addition, interleukin-3 (IL-3) and PMA were able to promote proliferation, an increase in Ca2þ concentration ([Ca2þ]i), and PKC activation in the IL-3- dependent stem cell line, FDCPMix 1 (Whetton et al., 1988a,b; Carroll and May, 1994; Musashi et al., 1997). PLCg is also known to participate in lymphopoiesis (Hashimoto et al., 2000; Wang et al., 2000; Serrano et al., 2005). Whereas the PLCg1 isoform is predominantly expressed in T cells (Serrano et al., 2005), PLCg2 is expressed at high levels in B cells (Hashimoto et al., 2000; Wang et al., 2000). Recently, we have demonstrated the role of Ca2þ signaling in myeloid proliferation and differentiation of hematopoietic progenitor cells. A modest increase in [Ca2þ]i by IL-3 and GM-CSF was associated with increased hematopoietic stem/progenitor cell proliferation and participation of gap junctions. In contrast, large increases in [Ca2þ]i stimulated by ATP and its analogs promoted weak proliferation and induced differentiation of hematopoietic cells in macrophages without the involvement of gap junctions (Paredes-Gamero et al., 2008). However, the PLCg isoform related with the myeloid population remains unknown.
Cytokines are a group of molecules that couple with their receptors and trigger many intracellular signaling pathways, including those that control hematopoiesis (Smithgall, 1998; Zhu and Emerson, 2002). The binding of cytokines with their receptors induces a receptor dimerization and could induce the activation of a family of tyrosine kinases termed Janus kinases (JAK) depending on the particular cytokine receptor activated (Smithgall, 1998; Zhu and Emerson, 2002). IL-3 and granulocyte-macrophage colony-forming cells (GM-CFC), two important cytokines that regulate the hematopoiesis, activate JAKs, then phosphorylate receptors on target tyrosine residues that can serve as docking sites that allow the binding of other SH2-domain containing signaling molecules, such as signal transducers and activators of transcription (STATs), Src kinases, phosphatidylinositol 3-kinase (PI3K), PLCg, and other adaptor signaling proteins, such as Shc and Grb2 (Kirito et al., 1998; McCormack and Gonda, 2000; Geijsen et al., 2001; Lejeune et al., 2002; Wheadon et al., 2003; Van Meter et al., 2007). Downstream signal mediators, such as members of the mitogen-activated protein kinase (MAPK) families and Akt, further impart signal specificity in the hematopoietic system (Shelton et al., 2005; Van Meter et al., 2007; Geest and Coffer, 2009; Geest et al., 2009).
Several studies have described the interactions among classical Ca2þ-dependent kinases and pathways regulating cell survival and proliferation, such as the PI3K/Akt and Ras/Raf/ MAPK pathways (Agell et al., 2002; Minaguchi et al., 2006; Sindreu et al., 2007; Lopez-Alcala et al., 2008). However, in the hematopoietic system, how cytokine-stimulated Ca2þ signals converge on the MAPK pathway to alter proliferation and survival at the molecular level has not previously been defined.
In this report, multiparametric flow cytometry analysis was used to investigate the molecular mechanisms integrating Ca2þ signaling with the ERK pathway stimulated by IL-3 and GM-CSF in rare, enriched populations of murine and human hematopoietic stem/progenitor cells. We evaluated protein phosphorylation within a defined population that was enriched for murine (Lin—Sca-1þc-Kitþ: LSK) and human (CD45þCD34þCD38—) hematopoietic stem and progenitor cells. Our results show the involvement of PLCg2 in Ca2þ signaling elicited by IL-3 and GM-CSF as well as the participation of Ca2þ-dependent kinases in MEK activation in murine and human hematopoietic stem cell/progenitor populations.
Materials and Methods
Human and mouse bone marrow
The study was approved by the Ethics Committee of the Federal University of Sa˜o Paulo (mouse 1464/03; human 0600/05).Murine bone marrow cells were obtained from mice femur bones (C57Bl/6). Human bone marrow samples were collected after informed consent from healthy donors. These donors were admitted to the University Hospital (Hospital Sa˜o Paulo, Sa˜o Paulo, SP, Brazil) from 2005 to August 2009. The median age was 40 years, and ages ranged from 20 to 55 years.
Flow cytometry
Murine bone marrow cells were obtained by flushing with a physiological solution (137 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, 0.49 mM MgCl2, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM D-glucose). Human mononuclear fractions were obtained using Ficoll Hystopaque (1.077 g/cm3, Sigma Chemical Co.). Murine bone marrow cells (3 106 cells) and human mononuclear fractions (2 106 cells) were stimulated, respectively, with recombinant murine or human IL-3 (10 ng/ml) or GM-CSF (10 ng/ ml) at 378C in physiological solution (IL-3 and GM-CSF, Sigma). Cells were incubated for 1 h with inhibitors prior to stimulation with cytokines and then fixed with 2% paraformaldehyde (using concentrated FACS lysing solution, BD Biosciences) for 30 min.
The cells were washed with 0.1 M glycine, permeabilized (for intracellular labeling) with 0.01% saponin (p-MEK1/2[Ser218/ Ser222], p-PLCg, p-PLCg1[Tyr783], p-PLCg2[Tyr759], p-CaMKII[Thr286], p-PKCpan), or 0.001% Triton (p-ERK1/ 2[Thr202/Tyr204] for 15 min, and washed in PBS. Subsequently, the cells were incubated for 2 h with rabbit anti-p-PKCpan, anti-p-CaMKII, anti-p-PLCg1, and anti-p-PLCg2 (4 mg/ml, Cell Signaling Technology) or goat anti-p-PLCgpan (2 mg/ml, Becton Dickinson) or anti-p-MEK1/2 (4 mg/ml, Santa Cruz Biotechnology). All antibodies were diluted in PBS with 1% albumin. Appropriate rabbit or goat anti-IgG-Alexa Fluor 488-conjugated antibodies (Invitrogen/Molecular Probes) were incubated for 40 min as secondary antibodies.
To identify murine progenitor cells, a biotin-conjugated lineage (Lin) antibody cocktail was used (anti-Gr-1, Mac-1, CD3e, TER119, and B220) for 20 min, followed by labeling with 0.15 mg/ml streptavidin-PerCP, 0.1 mg/ml anti-stem cell Ag (Sca)-1-PE, and 0.15 mg/ml anti-c-Kit-APC. To identify human progenitor cells, after fixation and labeling with signaling antibodies, mononuclear cells were labeled with 5 ml CD45-PerCP, 5 ml CD34-APC, and 5 ml CD38-PE. All these antibodies were purchased from Becton Dickinson.
A total of 300,000 events were acquired per murine or human samples. Forward and side light scatter gates were used to exclude dead cells and debris. An argon laser was used for fluorescence excitation of Alexa Fluor 488, PE, and PERCP at 488 nm, and a diode laser was used for fluorescence excitation of APC at 633 nm.
Negative controls were tested in the absence of primary antibody. Compensation was performed using the samples. Data analyses were performed on a FACSCalibur (Becton Dickinson) flow cytometer using the CellQuest software (Becton Dickinson) and WinMDI 2.8 software available at http://facs.scripps.edu/ software.html.
Murine Lin—c-KitR and human CD34RFcR— isolation
Separation of murine Lin—c-Kitþ and human CD34þFcR— populations was performed by gradient centrifugation methods using Ficoll Hystopaque (1.077 g/cm3). After mononuclear isolation, the cells were labeled with specific antibodies. For murine cell isolation, biotin-conjugated Lin antibody cocktail was used, followed by incubation with anti-biotin-conjugated microbeads (Miltenyi Biotec). After labeling, the cells were placed in a magnetic column, and the negative population was extracted. The Lin— cells were labeled with anti-c-Kit-conjugated microbeads (Miltenyi Biotec) and the Lin—c-Kitþ population was isolated in a magnetic column. For human cell isolation, anti-CD34 and FcR blocking reagents were used (Miltenyi Biotec). The degree of cell purity was higher than 75% in the enriched populations.
Calcium measurements
For Ca2þ measurements in microtiter plates, 3 105 c-KitþLin— cells were placed per well in black 96-well microplates and loaded
with Fluo-4 Direct Calcium Assay Kit according to the manufacturer’s instructions (Molecular Probes/Invitrogen). After 1 h of the indicator incorporation at 378C, the cells were centrifuged and the fluorescence determined in a FlexStation 3 microplate reader (Molecular Devices). The Fluo-4 was excited at 490 nm, and light emission was detected at 525 nm.
For Ca2þ measurements by confocal microscopy, cytospins equal volume of 2 sodium dodecyl sulfate (SDS) gel loading buffer [100 mM Tris–HCl (pH 6.8), 200 mM dithiothreitol (DTT), 4% SDS, 0.1% bromophenol blue, and 20% glycerol] was added to lysates, which were subsequently heated at 1008C for 5 min. From each sample, 50 mg of protein was run on an SDS–PAGE gel and blotted onto a PVDF membrane (Millipore). The blots were blocked with 2% fat-free dried milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) for 1 h at room temperature before overnight incubation at 48C with primary antibody (b-actin, p-MEK1/ 2[Ser218/Ser222], p-PLCg, p-PLCg1[Tyr783], and p-slides (25 mm). The cells were incubated at room temperature for 40 min with 10 mM Fluo-4/AM and 0.01% pluronic acid and washed with physiological solution. Images were captured with a microscope (Zeiss, Axiovert 100 M, Germany) equipped with a laser scanner (Zeiss, LSM 510 META) and a 63 objective (Plan-Neofluor, 1.4 numerical aperture) under oil immersion. The Fluo-4 probe was excited with an argon laser (lEx 488 nm) and light emission was detected using a Zeiss META detector (lEm 500–550 nm). The pinhole device was not used for Ca2þ measurements. Images were collected at approximately 4–6 sec intervals. Fluorescence intensity was normalized with reference to the basal fluorescence using Examiner 3.2 (Zeiss) and Spectralyzer software.
IP3 formation assay
The human and mouse hematopoietic stem/progenitor cells were washed twice with PBS/EDTA (137 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 1.76 mM KH2PO4, and 0.5 mM EDTA, pH 7.4) and resuspended in Ca2þ and Mg2þ free PBS. In a 96-well black polypropylene plate (Nalge Nunc International), 20 ml of samples containing 40,000 cells were added per well and stimulated with cytokines at room temperature according to the manufacturer’s instructions (HitHunter Inositol (1,4,5)-triphosphate [IP3] Assay, GE Healthcare). The reaction was interrupted after 30 sec with 10 ml perchloric acid (0.2 N) followed by the addition of 20 ml IP3 Tracer-Green reagent and 40 ml IP3 Binding Protein reagent. To capture the polarized signal of IP3 Tracer-Green binding to IP3 room temperature in HRP-conjugated secondary antibody diluted in blocking buffer. After a final 3 10 min wash in TBS, detection was performed by chemiluminescence (Pierce Biotechnology). Western blot analysis of the extracts from NIH/3T3 fibroblasts [American Type Culture Collection (ATCC)] untreated and PDGF-stimulated for 10 min was used as a positive control of PLCg1 activation using a p-PLCg1[Tyr783] antibody. As a positive control for PLCg2 activation, lysates from Jurkat cells (ATCC) were prepared 24 h after 2 Gy g-irradiation, and Western blot analysis was performed using a p-PLCg2[Tyr759] antibody. All phospho-specific and anti-rabbit HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology, the antibody against b-actin was from Santa Cruz Biotechnology, and the anti-goat HRP-conjugated secondary antibody was from Chemicon/Millipore.
Statistical analysis
The Fluo-4 fluorescence intensities were normalized with reference to basal intensity and were shown to be representative pseudocolored images according to a fluorescence intensity scale ranging from 0 (black) to 255 (white). Data were expressed as mean SEM. Statistical comparisons were performed by using Student’s t-test or variance analyses (ANOVA). Values of P < 0.05 were considered statistically significant. Results IL-3 and GM-CSF induce Ca2þ increase in murine Lin—c-Binding Protein, the probe was excited at 485 nm and light emission was detected at 530 nm in a FlexStation 3 microplate reader (Molecular Devices). This is a competitive binding assay, in which the IP3 produced by agonist stimulation displaces the IP3 Tracer- Green from the IP3 Binding Protein, increasing the rotation time of the tracer and decreasing the fluorescent polarized signal. Granulocyte-macrophage colony-forming units (GM-CFU) assay CFU assay was performed by plating 5 103 murine (Lin—c-Kitþ) and human (CD34þFcR) bone marrow cells in methylcellulose (Methocult 03434 or H4100; StemCell Technologies) supplemented with recombinant murine or human IL-3 or GM-CSF in 35-mm diameter dishes. The cells were cultured in a fully humidified air incubator with 5% CO2, at 378C, for 7 and 14 days for murine and human cells, respectively. At the end of the incubation period, colonies of more than 50 cells were counted using a dark field microscope. Western blotting Stimulation of bone marrow cells was terminated at the indicated time points by placing the cells on ice, centrifuging immediately, washing in ice-cold PBS, and resuspending the cell pellets in lysis buffer [50 mM Tris–HCl (pH 7.4), 1% Tween-20, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitors (1 mg/ml aprotinin, 10 mg/ml leupeptin and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride)] for 2 h on ice. Protein extracts were cleared by centrifugation, and protein concentration was measured using the RC DC protein assay kit (Bio-Rad, CA) according to the manufacturer’s instructions. An Fluo-4 was incorporated into isolated murine (Lin—c-Kitþ) and human (CD34þFcR—) progenitor cells. Figure 1A–C shows that cytokines induced a smaller Ca2þ increase. Both cytokines were able to induce proliferation and differentiation (Just et al.,1991; Domen and Weissman, 2000; McCormack and Gonda, 2000), although higher hematopoietic cell proliferation was achieved with the cytokine concentrations used in this study than previously reported (Paredes-Gamero et al., 2008). A sustained increase in Ca2þ was observed in the Lin—c-Kitþ population after IL-3 and GM-CSF stimulation (Fig. 1A,B). However, analysis by confocal microscopy indicates oscillations in Ca2þ concentration in response to cytokines (Fig. 1C).Overall, 50% of the murine hematopoietic stem/progenitor cells were responsive to IL-3 (total of 58 cells counted); similarly, approximately 49% of the cells were responsive to GM-CSF (total of 74 cells counted). Similar results were obtained in human CD34þFCR— cells (Fig. S1A,B). In human hematopoietic stem/progenitor cells, we found that 48% of cells were responsive either to IL-3 (total of 23 cells counted) or GM-CSF (total of 25 cells counted). Ca2þ measurement in an unstimulated sample is shown in the supplementary information (Fig. S1C). Furthermore, to corroborate the participation of intracellular Ca2þ stores, the levels of IP3 were measured. Cytokines were able to elevate IP3 concentrations in murine (Fig. 1D) and human hematopoietic stem/progenitor cells (Fig. S1D). To exclude the participation of extracellular Ca2þ in cytokine responses, Lin—c-Kitþ cells were stimulated in the presence of 10 mM EGTA; a Ca2þ chelator, and 1 mM nifedipine; an L-type voltage dependent Ca2þ channel blocker. The results showed that EGTA and nifedipine did not significantly abolish the Ca2þ increase elicited by cytokines (Fig. 1E). IL-3 and GM-CSF induce MEK and PLCg activation Evidence for MEK/ERK pathway activation in response to increasing [Ca2þ]i has been previously reported in other cell systems including hematopoietic cancer and lymphocytes (Agell et al., 2002; Sindreu et al., 2007; Lopez-Alcala et al., 2008; Roderick and Cook, 2008). Additionally, PLCg is a downstream effector of activated cytokine receptors and is involved in Ca2þ release. Therefore, we first determined the kinetics of PLCg and MEK activation in purified murine Lin—Sca1þc-Kitþ (or LSK) (Fig. 2A,B) and human CD45þCD34þCD38— (Fig. 3A,B) cells by flow cytometry. Murine LSK (Christensen and Weissman, 2001; Van Meter et al., 2007; Challen et al., 2009; Tarnok et al., 2010) and human CD45þCD34þCD38— (Sandhaus et al., 1998; Langenkamp et al., 2009; Tarnok et al., 2010) contain the highest numbers of hematopoietic stem and progenitor cells as described in several reports. PLCg and MEK were phosphorylated in murine (Fig. 2C) and human (Fig. 3C) hematopoietic stem/progenitor cells as a result of IL-3 and GM-CSF treatment. Moreover, activation of PLCg in these cells occurred within 5 min after stimulation and remained elevated for up to 30 min of evaluation (Figs. 2C and 3C), except for IL-3-stimulated murine hematopoietic stem/progenitor cells that showed an attenuated response after 15 min (Fig. 2C). Interestingly, while the magnitude and duration of MEK phosphorylation was similar in human hematopoietic stem/progenitor cells in spite of the stimulus used (Fig. 3C), different profiles of phospho-MEK were observed in the murine hematopoietic stem/progenitor cells after exposure to IL-3 and GM-CSF (Fig. 2C). In contrast to the sustained MEK activation observed in GM-CSF-stimulated cells, kinase phosphorylation was transient in the presence of IL-3. A time course evaluation of MEK and PLCg activation was also shown in whole murine bone marrow by Western blot analysis, due to the low frequency of LSK cells available to immunoblot. Stimulation of total bone marrow cells with 10 ng/ ml IL-3 resulted in a time-dependent increase of MEK and PLCg phosphorylation (Fig. S2), corroborating previous reports showing that analysis of the whole bone marrow cannot discriminate among specific responses elicited by hematopoietic cell lineages (Krutzik and Nolan, 2003; Van Meter et al., 2007). Fig. 2. IL-3 and GM-CSF induce PLCg and MEK1/2 activation in murine hematopoietic stem/progenitor cells. A,B: The region of murine hematopoieticstem/progenitorcellsanalyzedisshown. A: Theregionlineage negative(Lin—) wasgated, andinthis region,(B) thephosphorylation of proteins in the LSK population was assessed. C: Temporal phosphorylation of (a and b) MEK1/2 and (c and d) PLCg was evaluated in LSK population. Filled and open histograms represent unstimulated and stimulated samples, respectively. Data are representative of at least five experiments. Fig. 3. IL-3 and GM-CSF induce PLCg and MEK1/2 activation in human hematopoietic stem/progenitor cells. A,B: The region of human hematopoietic stem/progenitor cells analyzed is shown. In the region gated (A), the phosphorylation of proteins (B) in the CD45RCD34RCD38— population was assessed. C: Temporal phosphorylation of (a and b) MEK1/2 and (c and d) PLCg was evaluated in CD45RCD34RCD38— cells. Filled and open histograms represent unstimulated and stimulated samples, respectively. Data are representative of at least four experiments. Ca2þ release induces PKC and CaMKII-dependent MEK phosphorylation As demonstrated above, IL-3 and GM-CSF binding to receptors resulted in PLCg activation, which can induce Ca2þ release in both murine and human hematopoietic stem/progenitor cells. If MEK activation is dependent on Ca2þ signaling, its activation will be sensitive to Ca2þ signaling inhibitors. To verify this hypothesis, cells were incubated for 1 h with the Ca2þ signaling inhibitors 2-APB (IP3 receptor/channel antagonist and store operated Ca2þ channels), GF109203 (PKC inhibitor), and KN-62 (CaMKII inhibitor) before stimulation with cytokines. The MEK inhibitor PD98059 was used as a negative control (Alessi et al., 1995; Pang et al., 1995; Yang et al., 1999; Lunghi et al., 2003; Nakata, 2004). As can be seen in Figure 4A (murine hematopoietic stem/progenitor cells) and Figure 4B (human hematopoietic stem/progenitor cells), MEK phosphorylation was partially inhibited by 2-APB, KN-62, and GF109203. Statistical analysis of MEK phosphorylation by cytokines is shown in Figure S3. Similar results were obtained for human and mouse hematopoietic stem/progenitor cells. These data are consistent with the idea that activation of PLCg in response to IL-3 and GM-CSF can induce the release of Ca2þ from the endoplasmic reticulum stores by opening the IP3 receptor/channel, which, in turn, activates PKC and CaMKII. We next asked whether inhibition of ERK1/2 signaling using the MEK inhibitor PD98059 would affect PLCg activation. As a negative control, the PLC inhibitor U73122 was used. IL-3 and GM-CSF-induced PLCg activation was not influenced by MEK inhibition (Fig. 5). As expected, the PLCg inhibitor U73122 abolished protein activation. These results further indicate that the MEK activation can be modulated or is partially dependent on PLCg signal. In addition, the activation of ERK1/2 by IL-3 and GM-CSF was corroborated in mouse and human hematopoietic stem/ progenitor cells. ERK1/2 was strongly phosphorylated in the LSK population by IL-3, but unexpectedly only a mild phosphorylation was caused by GM-CSF (Fig. 6A). Moreover, human ERK1/2 was only moderately activated after a 5-min stimulation with IL-3 and GM-CSF (Fig. 6B). Statistical evaluation of ERK1/2 phosphorylation is shown in Figure S4.Figure S5 shows ERK1/2 nuclear translocation in c-KitþLin— population after stimulation with both cytokines, as evaluated by confocal microscopy. IL-3 and GM-CSF promote their effects via PLCg2 activation To determine the subtype of PLCg activated in murine and human hematopoietic stem/progenitor cells by IL-3 and GM-CSF stimulation, specific antibodies against p-PLCg1 and p-PLCg2 were used. Neither IL-3 nor GM-CSF was able to activate PLCg1 in murine and human hematopoietic stem/ progenitor cells (Fig. 7A,B), although in one of four healthy donors PLCg1 phosphorylation was detected in human hematopoietic stem/progenitor cells after treatment with cytokines (data not shown). In contrast, activation of PLCg2 was induced by the stimulation of murine (Fig. 7A) and human hematopoietic stem/progenitor cells (Fig. 7B) with both cytokines. In agreement with this idea, we found an increased phosphorylation of PLCg2 in total murine bone marrow cells stimulated with 10 ng/ml IL-3 by Western blot analysis (Fig. 7C). Moreover, positive controls for phospho-PLCg1 and phospho- PLCg2 antibody specificities were demonstrated by Western blot analysis of lysates from NIH/3T3 fibroblasts treated with PDGF for 10 min (Fig. 7D) (Kim et al., 1991) and g-irradiated- Jurkat T cells (Fig. 7E) (Park et al., 2002), respectively. Statistical evaluation of PLCg phosphorylation is shown in Figure S6. The functional importance of PLCg2 in human and murine myelopoiesis was subsequently investigated using the CFU assay. Murine Lin—c-Kitþ and human CD34þFcR— cell fractions enriched with hematopoietic stem and progenitor cells were isolated and cultivated in methylcellulose supplemented with IL-3 or GM-CSF in the presence and absence of the PLCg inhibitor (U73122). The growth of murine and human GM-CFU was markedly reduced in the presence of U73122. As can be seen in Figure 8A,B, the numbers of murine and human GM-CFU stimulated by IL-3 were reduced approximately 25% and 40%, respectively. A similar inhibitory effect was observed in murine and human GM-CFU stimulated with GM-CSF, reaching levels 40% below controls in the presence of U73122 (Fig. 8A,B). Taken together, these biochemical and cellular assays indicate a requirement for PLCg2 in IL-3 and GM-CSF- stimulated myeloid differentiation of hematopoietic stem/ progenitor cells. Discussion Ca2þ is a ubiquitous messenger that controls many cellular physiological processes such as proliferation, differentiation, and cell death. The involvement of Ca2þ signaling in cytokine- mediated signaling has been previously reported (Whetton et al., 1988a; Ren et al., 1994; Collison et al., 1998; Dutt et al., 1998; Tong et al., 2004; Paredes-Gamero et al., 2008). However, in these studies the interaction between Ca2þ signaling and other important intracellular pathways in hematopoiesis in undifferentiated stem/progenitor-enriched fractions has not been addressed. Previously, we found that several cytokines that induce different responses in the hematopoietic system such as IL-3, IL-6, IL-7, stem cell factor, granulocyte-CSF, macrophage-CSF, GM-CSF, and erythropoietin are all able to stimulate a Ca2þ increase in hematopoietic progenitor cells (Paredes-Gamero et al., 2008). Other reports have also shown a role for of Ca2þ signaling in mature cells such as lymphocytes (Hashimoto et al., 2000; Wilde and Watson, 2001; Harnett et al., 2005; Serrano et al., 2005). Despite these key points, almost nothing is known about the molecular mechanisms evoked by Ca2þ in cytokine- stimulated signaling in murine and human hematopoietic stem/progenitor cells. To address this issue, we applied multiparametric flow cytometry analysis to investigate the molecular mechanisms integrating Ca2þ signaling with the ERK pathway in primary murine and human hematopoietic stem/progenitor cells stimulated with IL-3 and GM-CSF. It has been previously demonstrated that the release of Ca2þ by cytokines is associated with the activation of PLCg, which recognizes phosphorylated tyrosine residues in cytokine receptors (Whetton et al., 1988a; Ren et al., 1994; Collison et al., 1998; Dutt et al., 1998; Hashimoto et al., 2000; Ueda et al., 2002; Tong et al., 2004; Serrano et al., 2005; Paredes-Gamero et al., 2008). In lymphocytes, PLCg1 isoform is related to T cell-antigen receptor signaling (Serrano et al., 2005), and PLCg2 is predominantly expressed in B lymphocytes and is associated with B cell-antigen receptor signaling (Hashimoto et al., 2000; Wang et al., 2000). In this study, we showed that IL-3 and GM-CSF induce Ca2þ release in murine and human hematopoietic stem/progenitor cells and that this event was associated with PLCg2 activation (Fig. 7). Therefore, the PLCg2 subtype is related to myeloid differentiation. The interaction between PLCg/Ca2þ signaling and the Ras/ Raf/MEK/ERK pathway has been described in different models including hematopoietic cancer and lymphocytes (Agell et al., 2002; Sindreu et al., 2007; Lopez-Alcala et al., 2008; Roderick and Cook, 2008). Ca2þ-dependent proteins, such as PKC and CaMKII, are associated with these effects in several cell types (Valledor et al., 1999; Choe and Wang, 2002; Illario et al., 2005; Kudirka et al., 2007; Montiel et al., 2007). These studies show that inhibition of Ca2þ-kinase dependent proteins negatively modulate the activation of the MEK/ERK pathway, suggesting that PKC, CaMKII, and probably other Ca2þ-dependent proteins are key intermediate modulators in this cascade. In agreement with these observations, our results show that the pharmacological inhibitors of PKC (GF109203) and CaMKII (KN-62), and other Ca2þ signaling inhibitors negatively modulate IL-3 and GM-CSF-induced MEK activation in murine and human hematopoietic stem/progenitor cells (Fig. 4). Our findings suggest that IL-3 and GM-CSF induce a specific Ca2þ signal that modulates the MEK/ERK cascade through Ca2þ-dependent kinases. In addition, in the hematopoietic system, participation of Ca2þ-dependent kinases have been previously documented in primitive hematopoietic lineages (Whetton et al., 1988a,b; Carroll and May, 1994; Musashi et al., 1997). IL-3 and PMA induce PKC activation and cell proliferation (Whetton et al., 1988a,b; Carroll and May, 1994; Musashi et al., 1997), and an increase in Ca2þ is observed in the cytokine-dependent multipotent stem cell line, FDCP-Mix 1 (Whetton et al., 1988a). Furthermore, CaMKIV was found to be necessary for the maintenance of the quiescent state of hematopoietic stem/ progenitor cells (Kitsos et al., 2005). PKC activation by IL-3 was also found in long-term bone marrow cultures, whereas GM-CSF was not able to activate this kinase (Paredes-Gamero et al., 2008).
The MEK/ERK pathway is one of the most important intracellular pathways investigated in the hematopoietic system (Wojchowski et al., 1999; Shelton et al., 2005; Van Meter et al., 2007; Geest and Coffer, 2009; Geest et al., 2009). IL-3 and GM-CSF binding to receptors results in MEK1/2 and ERK1/2 phosphorylation in both murine and human hematopoietic stem/progenitor cells (Figs. 2 and 3). Differences in the kinetics of MEK phosphorylation induced by these cytokines were evident in murine LSK cells because a transient response to IL-3 and a sustained activation in the presence of GM-CSF were observed. ERK1/2 also displayed major differences in phosphorylation depending on the stimulus. In parallel to the change in phosphorylated MEK levels, murine LSK cells stimulated by IL-3 had robust ERK activation (Fig. 6A). In contrast, the lower levels of ERK phosphorylation in response to GM-CSF in murine LSK cells, as well as in human hematopoietic stem/progenitor cells stimulated with IL-3 and GM-CSF (Fig. 6), could be regulated by the sustained MEK activation observed in these situations (Figs. 2 and 3).
It was hypothesized that each type of cell expresses a particular pattern of components from the Ca2þ signaling pathway, thereby generating a particular magnitude, time course, and intracellular location (Berridge, 2006; Cook and Lockyer, 2006; Rizzuto and Pozzan, 2006). This Ca2þ fingerprint encodes information that allows Ca2þ signaling to control diverse cellular processes in a specific manner (Roderick and Cook, 2008). There are many proteins that participate in translating the Ca2þ signaling; among them the classical PKC and CaM, that activate CaMKs, are the best characterized.
In addition, PLCg activation also can regulate Ras and diacylglycerol regulates guanine nucleotide exchange factors and proteins, such as calcineurin, CaMKII and PKC, which act in the progression of the cell cycle (Cullen and Lockyer, 2002; Bivona et al., 2003; Zheng et al., 2005; Roderick and Cook, 2008). The irregular increase in Ca2þ signaling after stimulation with cytokines (Fig. 1C) is in agreement with our previous report in the primitive hematopoietic cells of bone marrow cultures (Paredes-Gamero et al., 2008). This oscillatory response observed in Ca2þ could be related to the participation of proteins such as RASAL that recognizes oscillatory Ca2þ variations (Liu et al., 2005), or PLCe that is regulated by direct binding of small G-proteins such as Ras (Kelley et al., 2001; Oestreich et al., 2009). The investigation of oscillatory events in hematopoietic cells and the activation of these novel Ca2þ-sensitive proteins certainly deserve further attention.
In this study, similar responses were observed between human and murine hematopoietic cells. IL-3 and GM-CSF are important cytokines that participate in the proliferation and differentiation of the myeloid lineage (Just et al., 1991; Domen and Weissman, 2000; McCormack and Gonda, 2000). The concentrations of cytokines used in this study are known to induce a high index of hematopoietic proliferation but at the same time, differentiation occurs without a decrease in the progenitor pull (Paredes-Gamero et al., 2008). These cytokines bind similar receptors, each of them consisting of a cytokine specific a-chain and a b-chain common to all receptors of this family. In mice, a second b-chain has been identified that associates exclusively with the IL-3 a-chain (Geijsen et al., 2001). Interestingly, the human IL-3 receptor response, and human and murine GM-CSF receptor responses showed similar patterns of intracellular protein activation: sustained activation of PLCg2 and MEK and poor ERK activation with a great dependence on PLC activation to induce GM-CFU formation. On the other hand, mouse IL-3 receptor response showed transient PLCg2 and MEK activation, and ERK activation with less dependence on PLC activation to induce GM-CFU. Therefore, the particular responses of murine and human hematopoietic stem/progenitor cells to these cytokines might reflect differences in IL-3 and GM-CSF binding to specific receptors, besides the magnitude and time course of downstream signaling.
In conclusion, we showed that activation of Ca2þ signaling by the myeloid cytokines, IL-3 and GM-CSF, occurs in primary human and murine hematopoietic stem/progenitor cells through the activation of PLCg2 (Fig. 9). The importance of PLCg2 in other myeloid stimulators are under investigation. Moreover, Ca2þ signaling induces the activation of kinases, such as PKC and CaMKII, that can modulate the MEK/ERK pathway (Fig. 9). Finally, we showed that Ca2þ and MEK/ERK signaling mechanisms are conserved in human and murine cells. The many options for downstream signaling modulated by the Ca2þ pathway orchestrate the proliferation and differentiation of hematopoietic precursors. The deregulation of these specific mechanisms may contribute to proliferative disorders, such as cancer, including disorders in hematopoietic cells as Ca2þ- dependent signaling mechanisms are frequently remodeled or deregulated in cancer cells (Agell et al., 2002; Cook and Lockyer, 2006).