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. Electronic supplementary material The online version of this article (doi:10.1007/s12015-016-9689-x) contains supplementary material, which is available to authorized users.

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 [5]. 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 [1], its contrasting migration-promoting role for T lymphocytes was demonstrated in more recent work [18]. As of today, the overall consensus is that PLC signaling does not inhibit [1] but instead promotes cell trafficking [18].
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 [19].

Animals
Pathogen-free, 4-6-week-old C57BL/6 J wild-type mice (WT) and B6.129S1-Plcβ2 tm1Dwu /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 [21].

Fibronectin Cell-Adhesion Assay
Murine BMMNCs were resuspended in RPMI 1640 plus 0.5 % bovine serum albumin (BSA) medium (5 × 10 4 cells/ 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 × 10 6 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 granulocytemacrophage 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 % CO 2 ) 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 γirradiation (lethal dose). After 24 h, mice were transplanted with 1.5 × 10 5 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 % CO 2 ) 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 × 10 5 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].

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 β2microglobulin 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).

Western Blotting
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].

Statistical Analysis
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.
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.
First, we labeled BMMNCs from PLC-β2-KO and WT animals with the cell membrane-binding fluorochrome PKH67 and injected them into lethally irradiated syngeneic WT animals. Figure 2a shows that, 24 h after transplantation, cells from PLC-β2-KO mice homed to BM in lower numbers than WT cells. Next, we transplanted lethally irradiated syngeneic animals with BMMNCs from mutant and WT mice, and 12 days later we evaluated the number of spleen colonies formed by the early progenitor cells known as colony-forming units in spleen (CFU-S) as well as the number of clonogenic CFU-GM progenitors present in the BM of transplanted animals. Figure 2b again shows a decrease in early engraftment of PLC-β2-KO mouse-derived HSPCs. Finally, we analyzed the recovery of peripheral blood counts in lethally irradiated mice transplanted with PLC-β2 and WT cells and observed a delayed recovery of leucocytes and platelet counts (Fig. 2c) in mice transplanted with HSPCs from PLC-β2-deficient mice.

Defective Homing and Engraftment of Mutant HSPCS Is Explained by Changes in PLC-β2-Mediated Signaling
In our previous paper we already demonstrated that BMderived cells from PLC-β2 mutant mice have impaired phosphorylation of p42/44 MAPK and AKT after stimulation with mobilization-promoting factors [5]. Therefore, since PLC-β2 is involved in the release of calcium from intracellular stores, we evaluated the intracellular level of Ca 2+ in BM-sorted Sca-1 + cells enriched for HSPCs (Fig. 3a) or Gr-1+ cells (Fig. 3b) from mutant and WT mice in response to the crucial homing factors SDF-1 and S1P. As a positive control, we also stimulated these cells with C5a and desArg C5a, which are known to be potent stimulatory factors for these cells. As expected, we observed impaired Ca 2+ release from the mutant cells.
Finally, since we recently reported that the chemotaxis of HSPCs is negatively regulated by heme oxygenase 1 (HO-1) [19], 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.

Discussion
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.
Members of the PLC family play an important role in inducing two second messengers, diacylglycerol (DAG) and inositol three phosphate (IP 3 ), in cells [5] (Fig. 4). These second messengers play an important role in activating phosphokinase C (PKC) and in the release of calcium from intracellular stores. It is known that the hematopoietic cell-specific isoform PLC-β2 is associated with several G protein-coupled receptors, including CXCR4, S1PR 1 , and P2Y receptors, which bind HSPC chemoattractants such as SDF-1, S1P, and The data in Panels a-c represent the combined results from two independent experiments (n = 10) for each panel. *p < 0.005 ATP, respectively [24,27]. In this migration process, an important role is played by the intracellular Ca 2+ level as well as by the activation of several downstream kinases, including p42/44 MAPK, AKT, and PKC-ζ [28]. In particular, PKC-ζ has been implicated in an elegant paper on SDF-1-CXCR4-mediated trafficking of human CD34 + HSPCs [29]. Moreover, in the same work injection of mice with inhibitory PKC-ζ pseudosubstrate peptides resulted in mobilization of HSPCs from BM into PB [29]. 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 BMretention of these cells [5]. 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 [29], 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.   Fig. 4 The role of PLC-β2 in homing of HSPCs. PLC-β2 is involved in signaling form all major homing receptors including CXCR4 (SDF-1 receptor), S1PR 1 (S1P receptor 1), purinergic P2X and P2Y (ATP receptors) and putative receptor for C1P (not cloned yet). Activation of PLC-β2 signaling is crucial for migration and adhesion of HSPCs in DAG -PI3K and IP-3 -Ca 2+ depended manner Since we already reported that PLC-β2 is involved in phosphorylation of p42/44 MAPK and AKT in murine BM cells [5], in the current work we studied its effect on Ca 2+ 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 Ca 2+ 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 [19], 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 [5], 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 [5]. 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 proadhesive 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 [1], 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 Figure 1) 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 [18].
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 [19]. 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.