The Involvment of Hematopoietic-Specific PLC -β2 in Homing and Engraftment of Hematopoietic Stem/Progenitor Cells
Migration and bone marrow (BM) homing of hematopoietic stem progenitor cells (HSPCs) is regulated by several signaling pathways, and here we provide evidence for the involvement in this process of hematopoietic-specific phospholipase C-β2 (PLC-β2). This enzyme is involved in release of intracellular calcium and activation of protein kinase C (PKC). Recently we reported that PLC-β2 promotes mobilization of HSPCs from BM into peripheral blood (PB), and this effect is mediated by the involvement of PLC-β2 in the release of proteolytic enzymes from granulocytes and its role in disintegration of membrane lipid rafts. Here we report that, besides the role of PLC-β2 in the release of HSPCs from BM niches, PLC-β2 regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including SDF-1, S1P, C1P, and ATP. Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release in PLC-β2-KO mice and a high baseline level of heme oxygenase 1 (HO-1), an enzyme that negatively regulates cell migration.
KeywordsPLC-β2 Stem cell homing HO-1 SDF-1 S1P C1P
The phospholipase C (PLC) family of enzymes consists of 13 members split between six subfamilies, including the PLC-δ (1, 3, 4), −β (1–4), −γ (1, 2), −ε, −ζ, and –η (1, 2) isoforms [1, 2, 3]. PLC enzymes are associated with cell surface receptors that convert phosphatidyloinositol-4,5-biphosphate into two important second messengers, diacylglycerol (DAG) and inositol-1,4.5-triphosphate (IP3) [3, 4, 5]. Among these isoforms, PLC-β2 is somewhat unique in being a hematopoietic-specific enzyme [1, 2].
Recently, we identified PLC-β2 as the first known lipolytic enzyme involved in the mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) [5, 6]. These pro-mobilizing effects depend on two important mechanisms. First, PLC-β2, as an intracellular enzyme involved in signaling from the receptor for the C5 cleavage fragment C5a (C5aR), promotes degranulation of granulocytes, which release proteolytic enzymes affecting cell adhesion-mediated retention mechanisms of HSPCs in their BM niches. These retention mechanisms involve the chemokine receptor CXCR4 and the very late antigen 4 receptor (VLA-4, also known as α4β1 integrin) expressed on the surface of HSPCs. Their respective ligands, the α-chemokine stromal cell-derived factor 1 (SDF-1) and vascular adhesion molecule 1 (VCAM-1, also known as CD106), are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts) [1, 6, 7, 8, 9, 10, 11]. Secondly, PLC-β2, when released extracellularly from granulocytes and HSPCs upon stimulation, cleaves the glycolipid glycosylphosphatidylinositol anchor (GPI-A) in cell membranes and thus disrupts the structure of membrane lipid rafts, which are important in the retention of HSPCs in BM niches [5, 6, 12]. It is well known that both BM-retention receptors for HSPCs, CXCR4, and VLA-4, are membrane lipid raft receptors [6, 13, 14, 15, 16, 17].
Taking into consideration the important role of PLC-β2 in promoting detachment of HSPCs from BM niches, it is not surprising that PLC-β2-KO mice are poor mobilizers . Nevertheless, while performing mobilization studies, we found that BM cells from these animals show somewhat reduced chemotaxis in response to several chemottractants involved in cell trafficking. Therefore, we became interested in the role of PLC-β2 in regulating the migration of HSPCs, as this enzyme is potentially involved in BM homing of HSPCs after transplantation.
However, in an initial old report describing PLC-β2 knockout mice, PLC-β2 was proposed to inhibit cell migration , its contrasting migration-promoting role for T lymphocytes was demonstrated in more recent work . As of today, the overall consensus is that PLC signaling does not inhibit  but instead promotes cell trafficking .
We report here that HSPCs from PLC-β2-KO mice show defective migration in response to BM-released chemoattractants and as result of this show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained, at least partially, by impaired calcium release and (phosphokinase C) PKC activation in PLC-β2-KO mice and an enhanced intercellular baseline level of the heme oxygenase 1 (HO-1) enzyme, which, as we recently reported, negatively regulates cell migration .
Material and Methods
Pathogen-free, 4–6-week-old C57BL/6 J wild-type mice (WT) and B6.129S1-Plcβ2tm1Dwu/J (PLC-β2-KO) female mice were purchased from the Jackson Laboratory (Bar Harbor, ME; USA) at least 2 weeks before experiments. Animal studies were approved by the Animal Care and Use Committee of the University of Louisville (Louisville, KY, USA) [5, 20].
Murine Bone Marrow-Derived Mononuclear Cells (BMMNCs)
BMMNCs were obtained by flushing tibias and femurs from WT and PLC-β2-KO mice. Red blood cells (RBCs) were removed by lysis in BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, USA), washed, and resuspended in appropriate media .
Sorting of Gr-1+ Cells
BM was flushed from the femurs and tibias of experimental mice, and after lysis of RBCs using 1 × BD Pharm Lyse buffer (BD Pharmingen, San Jose, CA, USA) the population of total nucleated cells was obtained. Cells were subsequently stained with antibodies: allophycocyanin (APC)–Cy7–anti-Ly6G (clone 1 A8), phycoerythrin (PE)–anti-CD11b (clone M1/70), and Alexa Fluor 488–anti-Ly6C (clone HK1.4) for 30 min in medium containing 2 % FBS (fetal bovine serum). The cells were then washed, resuspended in RPMI 1640 medium, and sorted using a Moflo XDP cell sorter (Beckman Coulter, Indianapolis, IN, USA) as populations of neutrophils (Ly6G+/CD11b+) [22, 23].
Transwell Migration Assay
BMMNCs and BM-derived Gr-1+ cells from WT and PLC-β2-KO mice were resuspended in assay medium (RPMI-1640 with 0.5 % BSA). Assay medium (650 μl), alone or containing stromal-derived growth factor 1 (SDF-1, 100 ng/ml), sphingosine-1-phosphate (S1P, 0.1 μM), ceramide-1-phosphate (C1P, 100 μM), or adenosine triphosphate (ATP, 0.25 μg/ml) for BMMNCs or SDF-1, chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES, 75 ng/ml), or chemokine (C-C motif) ligand 3 (CCL3, also known as MIP-1α, 10 ng/ml) for BM-derived Gr-1+ cells, was added to the lower chambers of a Costar Transwell 24-well plate (Corning Costar, Cambridge, MA, USA). Aliquots of cell suspension (1 × 106 cells per 100 μl) were loaded onto the upper chambers with 5-μm pore filters and then incubated for 3 h (37 °C, 5 % CO2). Aliquots of BMMNCs and Gr-1+ cells from the lower chambers were harvested and scored by FACS analysis. Briefly, the cells were gated according to their forward-scatter (FSC) and side-scatter (SSC) parameters and counted during a 30-s acquisition at a high flow rate. The rest of the BMMNCs recovered from the lower chamber were resuspended in human methylcellulose base medium provided by the manufacturer (R&D Systems), supplemented with murine GM-CSF (25 ng/ml) and IL-3 (10 ng/ml) for determining the number of CFU-GM colonies. Cultures were incubated for 7 days (37 °C, 95 % humidity, and 5 % CO2), at which time they were scored under an inverted microscope for the number of colonies [14, 19, 21].
Fibronectin Cell-Adhesion Assay
Murine BMMNCs were resuspended in RPMI 1640 plus 0.5 % bovine serum albumin (BSA) medium (5 × 104cells/100 μl). Subsequently, cell suspensions were incubated for 15 min at 37 °C, added directly to 96-well plates coated before the experiment with fibronectin (10 μg/ml), incubated overnight at 4 °C, and then blocked with medium containing 0.5 % BSA for 2 h at 37 °C. Non-adherent cells were then washed from the wells, and all adherent cells were counted using an inverted microscope [14, 19].
Short-Term Homing Experiments
C57BL/6 J (B6) mice were separated into two groups and irradiated with a lethal dose of γ-irradiation (1000 cGy). Twenty-four hours after irradiation, the animals were transplanted (by tail vein injection) with 4 × 106 BM cells from WT or PLC-β2-KO mice. Before transplantation cells were labeled with PKH67 Green Fluorescent Cell Linker (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s protocol. At 24 h after transplantation, BM cells from the femurs were isolated via Ficoll-Paque and divided. A part of the cells were analyzed on a flow cytometer. The rest of the cells were plated in serum-free methylcellulose cultures and stimulated to grow CFU-GM colonies with granulocyte-macrophage colony-stimulating factor (GM-CSF, 25 ng/ml) and interleukin 3 (IL-3, 10 ng/ml). After 7 days of incubation (37 °C, 95 % humidity, and 5 % CO2) the number of colonies was scored under an inverted microscope [19, 20].
Evaluation of Engraftment
For engraftment experiments, C57BL/6 J (B6) mice were divided into two groups and irradiated with 1000 cGy γ-irradiation (lethal dose). After 24 h, mice were transplanted with 1.5 × 105 BM cells from WT or PLC-β2-KO mice by tail-vein injection. Femora of transplanted mice were flushed with phosphate-buffered saline (PBS) on day 12 post transplantation. Purified via Ficoll-Paque, BM cells were plated in serum-free methylcellulose cultures and stimulated to grow CFU-GM colonies with G-CSF (25 ng/ml) plus IL-3 (10 ng/ml). After 7 days of incubation (37 °C, 95 % humidity, and 5 % CO2) the number of colonies was scored under an inverted microscope. Spleens from experimental mice were also removed, fixed in Telesyniczky’s solution for CFU-S assays, and the colonies on the surface of the spleen counted [19, 20].
Recovery of Leukocytes and Platelets
For transplantation experiments, mice were irradiated with a lethal dose of γ-irradiation (1000 cGy). After 24 h, the mice were transplanted by tail-vein injection with 2.5 × 105 BM cells. Transplanted mice were bled at various intervals from the retro-orbital plexus to obtain samples for white blood cell (WBC) and platelet (PLT) counts as described. Briefly, 50 μl of PB were taken into EDTA-coated Microvette tubes (Sarstedt Inc., Newton, NC, USA) and run within 2 h of collection on a HemaVet 950FS hematology analyzer (Drew Scientific Inc., Oxford, CT, USA) [19, 20].
Murine Sca-1+ enriched for HSPCs were isolated by immune-magnetic beads and Gr-1+ cells sorted as described  were resuspended in RPMI 1640 medium supplemented with 0.5 % BSA (2 × 106 cells per 400 μl medium) and incubated overnight at 37 °C. Subsequently, cells were stimulated by adding SDF-1 (100 ng/ml), S1P (0.1 μM), C5a (140 ng/ml), or desArgC5a (140 ng/ml) and incubated for 3 h at 37 °C. The cells were then centrifuged, and conditioned media (CM) were collected. Calcium levels were measured using the Calcium Quantification Assay kit (Abcam, Cambridge, MA, USA), according to the manufacturer’s protocol, by measuring the fluorescence in a fluorescence microplate reader (Beckman Coulter DTX 880 Multimode Detector, Beckman Coulter) using excitation at 540 nm and emission detection at 590 nm.
HO-1 Gene Expression by Real-Time Quantitative Reverse-Transcription PCR
Total RNA from WT and PLC-β2-KO mice was isolated from BM cells using RNeasy Kit (Qiagen, Valencia, CA) according to manufacturer’s protocol and then reverse-transcribed with MultiScribe reverse transcriptase and oligo-dT primers (Applied Biosystems, Foster City, CA). Quantitative assessment of mRNA levels was done by real-time qRT-PCR with Power SYBR Green PCR Master Mix reagent using ABI 7500 instrument. PCR conditions: 95 °C (15 s), 40 cycles at 95 °C (15 s), 60 °C (1 min). Only one PCR product was amplified, according to melting point analysis. The relative quantity of a target, normalized to the endogenous β2-microglobulin gene as control and relative to a calibrator, is expressed as 2–ΔΔCt (fold difference), where Ct is the threshold cycle, ΔCt = (Ct of target genes) – (Ct of the endogenous control gene, β2-microglobulin), and ΔΔCt = (ΔCt of samples for the target gene) – (ΔCt of the calibrator for the target gene) [14, 19]. Primer pairs used for the analysis: 5′-CCT CAC AGA TGG CGT CAC TT-3′ (forward) and 5′-GCT GAT CTG GGG TTT CCC TC-3′ (reverse).
To analyze the expression of HO-1, BMMNCs from WT and PLC-β2-deficient mice were harvested, centrifuged, and washed twice with PBS. Protein extracts were then obtained after cell lysis using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotechnology) and centrifugation at 15,000 rpm at −4 °C for 15 min. The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL) and Multimode Analysis Software (Beckman Coulter). Next, adjusted protein lysates (80 μg per sample) were separated on a 4–12 % SDS-PAGE gel, and fractionated proteins were transferred to a PVDF membrane (Bio-Rad). After blocking with 2.5 % non-fat dry milk in Tris-buffered saline containing 0.1 % Tween (TBST) for 1 h at room temperature, then washing with TBST, the membranes were incubated with a rabbit anti-HO-1 polyclonal antibody (Enzo Life Sciences, NY, USA, diluted 1:1000) overnight at 4 °C. Equal loading of proteins in all lanes was assured by reprobing with rabbit anti-β-actin monoclonal antibody (Novus Biologicals, USA, diluted 1:1000). Enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences) and film (Hyperfilm, Amersham Life Sciences) were used for band visualization [19, 21].
All results are presented as mean ± SD. Statistical analysis of the data was done using Student’s t-test for unpaired samples, with p ≤ 0.05 considered significant.
Bone Marrow Mononuclear Cells (BMMNCs) from PLC-β2-KO Mice Show an Impaired Chemotactic Response to BM Homing Factors
This decrease in chemotaxis was paralleled by increased spontaneous adhesion of BMMNCs to fibronectin-coated plates (Fig. 1b).
Bone Marrow Cells from PLCβ-2-KO Mice Show Defective Homing and Engraftment after Hematopoietic Transplantation
Since BMMNCs from PLC-β2-KO mice show defective in vitro migration in response to BM-secreted homing-relevant chemoattractants, we next moved to an in vivo model and employed three different assays.
Defective Homing and Engraftment of Mutant HSPCS Is Explained by Changes in PLC-β2-Mediated Signaling
Finally, since we recently reported that the chemotaxis of HSPCs is negatively regulated by heme oxygenase 1 (HO-1) , we evaluated the baseline level of HO-1 in WT and PLC-β2-KO BMMNC cells by RQ-PCR and western blotting (Fig. 3b) and found elevated HO-1 levels in mutant cells, which correlated with their impaired migratory potential.
The most important observation of this report is that hematopoietic-specific PLC-β2 enhances the responsiveness of HSPCs to BM-homing-related chemoattractants, including SDF-1, S1P, C1P, and ATP. By employing BM cells from PLC-β2-KO mice, we provided additional in vivo evidence for the role of this enzyme in directing homing and engraftment of HSPCs.
In our previous work we reported that PLC-β2-KO mice are poor mobilizers and explained this phenomenon by positing a requirement for this enzyme in the generation of a proteolytic and lipolytic microenvironment in BM following the degranulation of granulocytes. This microenvironment is responsible for the disintegration of adhesion interactions as well as integrity of membrane lipid rafts on the surface of HSPCs, which are important for BM-retention of these cells . Here we asked: what is the role of PLC-β2 in homing and engraftment of HSPCs after transplantation?
In our experiments we employed, in addition to SDF-1 , other chemotactic factors involved in HSPC homing, such as S1P, C1P, and extracellular ATP, and provided the first evidence that this enzyme regulates chemotactic responsiveness not only to SDF-1 but also other BM homing factors. We found that HSPCs from PLC-β2 mice have defective in vitro migration in response to BM homing factors in Transwell assays and in vivo show defective both homing and engraftment if transplanted into syngeneic WT animals.
Since we already reported that PLC-β2 is involved in phosphorylation of p42/44 MAPK and AKT in murine BM cells , in the current work we studied its effect on Ca2+ release and confirmed that stimulation of murine BM cells, not only by SDF-1 but also by S1P, C1P, and ATP, leads to increases in its intracellular level. It is well known that the intracellular Ca2+ level is regulated both spatially and temporally in cells and affects cell polarization, protrusion, retraction, and adhesion at the right place and time required for effective migration of HSPCs [30, 31]. This PLC-β2 effect is, as mentioned above, mediated in cells after activation of G protein-coupled receptors by specific ligands [3, 27].
Another interesting observation is the elevated steady-state level of HO-1 in PLC-β2-KO cells. As we have reported , HO-1 is a negative regulator of cell migration and, based on the results reported in this paper, its level may be regulated by PLC-β2. In support of this possibility, HSPCs from mutant animals showed impaired migratory responsiveness to several chemotactic factors. These effects correlate with a decrease in the release of calcium from intracellular stores, a decrease in phosphorylation of AKT and p42/44 MAPK , and an increase in HO-1 expression.
Our results reported here also have important implications for inhibiting cancer metastasis, e.g., to BM. Based on our results we argue that inhibition of PLC by specific inhibitors may decrease the migration of cancer cells and thus their metastatic potential. In fact, small molecule inhibitors of this enzyme have already been developed and tested in in vivo animal models [32, 33].
Moreover, based on our previously published results, HSPCs from PLC-β2-KO mice should have more stable membrane lipid rafts . This would explain their enhanced adhesion to fibronectin-coated plates compared with cells from WT control animals. Lipid rafts, however, also play an important role in cell migration by facilitating better contact of the G protein-coupled cell surface receptors CXCR4 and VLA-4 with downstream signaling molecules [5, 6, 12]. In the case of PLC-β2-KO HSPCs, it seems that their pro-adhesive properties are less affected than their pro-chemotactic responsiveness, because, in contrast to adhesion, chemotaxis in these cells is significantly impaired.
Interestingly, in an initial paper published two decades ago that described PLC-β2-KO mice, it was reported that BM cells from these animals had enhanced chemotaxis in response to chemokines, including RANTES and IL-8 , which suggested an inhibitory effect of this enzyme on cell trafficking that somewhat contradicted our results. Therefore, we evaluated the responsiveness of BMMNCs to RANTES and IL-8 gradients (Supplementary Figure1) and found that, like the other chemoattractants tested in our study, BMMNCs showed impaired migration in response to these chemokines. Moreover, a defect in migration in PLC-β2-KO cells was observed for other chemotactic factors as well. In corroboration of our observations, PLC-β2 has been confirmed to be required for proper migration of T cells in other more recent papers .
In conclusion, we demonstrated for the first time that PLC-β2 signaling is required for proper migratory responsiveness to all known BM-homing chemotactic factors and plays a role in the engraftment of HSPCs. Since in our in vivo hematopoietic reconstitution studies (Fig. 2) we evaluated short term repopulating hematopoietic stem cells (HSCs), in a future serial transplants experiments are needed to assess the compartment of more primitive long tem repopulating HSCs. At the molecular level, this defect described herein in PLC-β2-KO HSPCs depends on the impaired activation of signaling pathways involved in stem cell migration and adhesion and correlates with elevated baseline levels of heme oxygenase 1 (HO-1), an enzyme that is a negative regulator of cell migration . However, since wild type mice still engraft with BMMNC from PLC-β2-KO our data indicates that there are some PLC-β2-independent redundant mechanism that govern homing and engraftment. This is most likely explained by a fact that trafficking of HSPCs, as a developmental ancient physiological mechanism, has to be well protected from potential single gene mutation.
Supported by NIH grant R01 CA106281, NIH R01 DK074720, and the Stella and Henry Hoenig Endowment to MZR. Dr. Abdel-Latif is supported by the University of Kentucky Clinical and Translational Science Pilot Award (UL1TR000117), the UK COBRE Early Career Program (P20 GM103527), and NIH Grant R56 HL124266. AAI was on leave of absence from Faculty of Vet. Med. Zagazig Univ., Egypt.
Compliance with Ethical Standards
Conflict of Interest
The authors declare no potential conflicts of interest.
- 5.Adamiak, M., Poniewierska-Baran, A., Borkowska, S., et al. (2016). Evidence that a lipolytic enzyme-hematopoietic-specific phospholipase C-β2-promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes. Leukemia, 30(4), 919–928.CrossRefPubMedGoogle Scholar
- 21.Ratajczak, M. Z., Lee, H., Wysoczynski, M., et al. (2010). Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex. Leukemia, 24(5), 976–985.CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Ratajczak, M. Z., Kim, C. H., Janowska-Wieczorek, A., & Ratajczak, J. (2012). The expanding family of bone marrow homing factors for hematopoietic stem cells: stromal derived factor 1 is not the only player in the game. Scientific World Journal, 2012, 758512.CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Hou, C., Kirchner, T., Singer, M., Matheis, M., Argentieri, D., & Cavender, D. (2004). Vivo activity of a phospholipase C inhibitor, 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), in acute and chronic inflammatory reactions. The Journal of Pharmacology and Experimental Therapeutics, 309(2), 697–704.CrossRefPubMedGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.