PRL2/PTP4A2 Phosphatase Is Important for Hematopoietic Stem Cell Self-Renewal

Authors

  • Michihiro Kobayashi,

    1. Department of Pediatrics, Herman B Wells Center for Pediatric Research and Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Yunpeng Bai,

    1. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Yuanshu Dong,

    1. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Hao Yu,

    1. Department of Pediatrics, Herman B Wells Center for Pediatric Research and Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Sisi Chen,

    1. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Rui Gao,

    1. Department of Pediatrics, Herman B Wells Center for Pediatric Research and Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Lujuan Zhang,

    1. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Mervin C. Yoder,

    1. Department of Pediatrics, Herman B Wells Center for Pediatric Research and Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Reuben Kapur,

    1. Department of Pediatrics, Herman B Wells Center for Pediatric Research and Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Zhong-Yin Zhang,

    Corresponding author
    1. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    • Correspondence: Zhong-Yin Zhang, Ph.D., Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. e-mail: zyzhang@iu.edu; or Yan Liu, Ph.D., Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. e-mail: liu219@iu.edu

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  • Yan Liu

    Corresponding author
    1. Department of Pediatrics, Herman B Wells Center for Pediatric Research and Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    • Correspondence: Zhong-Yin Zhang, Ph.D., Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. e-mail: zyzhang@iu.edu; or Yan Liu, Ph.D., Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. e-mail: liu219@iu.edu

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Abstract

Hematopoietic stem cell (HSC) self-renewal is tightly controlled by cytokines and other signals in the microenvironment. While stem cell factor (SCF) is an early acting cytokine that activates the receptor tyrosine kinase KIT and promotes HSC maintenance, how SCF/KIT signaling is regulated in HSCs is poorly understood. The protein tyrosine phosphatase 4A (PTP4A) family (aka PRL [phosphatase of regenerating liver] phosphatases), consisting of PTP4A1/PRL1, PTP4A2/PRL2, and PTP4A3/PRL3, represents an intriguing group of phosphatases implicated in cell proliferation and tumorigenesis. However, the role of PTP4A in hematopoiesis remains elusive. To define the role of PTP4A in hematopoiesis, we analyzed HSC behavior in Ptp4a2 (Prl2) deficient mice. We found that Ptp4a2 deficiency impairs HSC self-renewal as revealed by serial bone marrow transplantation assays. Moreover, we observed that Ptp4a2 null hematopoietic stem and progenitor cells (HSPCs) are more quiescent and show reduced activation of the AKT and ERK signaling. Importantly, we discovered that the ability of PTP4A2 to enhance HSPC proliferation and activation of AKT and ERK signaling depends on its phosphatase activity. Furthermore, we found that PTP4A2 is important for SCF-mediated HSPC proliferation and loss of Ptp4a2 decreased the ability of oncogenic KIT/D814V mutant in promoting hematopoietic progenitor cell proliferation. Thus, PTP4A2 plays critical roles in regulating HSC self-renewal and mediating SCF/KIT signaling. Stem Cells 2014;32:1956–1967

Introduction

In order to maintain hematopoietic homeostasis throughout the life of an animal, the hematopoietic stem cell (HSC) pool must be maintained through the process of self-renewal [1]. HSC self-renewal requires the integration of survival and proliferation signals to maintain an undifferentiated state. This demands a complex crosstalk between extrinsic signals from the microenvironment and the cell-intrinsic regulators of HSCs [2, 3]. Stem cell factor (SCF) is a dimeric molecule that exerts its biological functions by binding to and activating the receptor tyrosine kinase KIT [4]. KIT is highly expressed in HSCs and SCF/KIT signaling plays a critical role in HSC maintenance [5–14]. SCF is an early acting cytokine that promotes HSC proliferation and survival [5–8]. Studies in mice with either partial or complete loss-of-function KIT mutations have revealed severe hematopoietic deficiencies of multiple lineages as well as an important role of KIT in sustaining rapidly cycling HSCs post-transplantation when competing with wild-type HSCs [9–14]. Despite the wealth of knowledge on SCF/KIT signaling, it is poorly understood how KIT signaling is regulated in HSCs [4].

Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors, systemic mastocytosis, and acute myelogenous leukemia (AML) [15–17]. These mutations results in altered substrate recognition and constitutive tyrosine autophosphorylation leading to promiscuous and constitutive signaling [18–20]. Consequently, cell lines and primary bone marrow (BM) cells that express the oncogenic KIT mutant demonstrate ligand-independent proliferation in vitro and myeloproliferative disease (MPD) in vivo [18–22]. However, the intracellular signals that contribute to mutant KIT-induced MPD are not known.

The PRL (phosphatase of regenerating liver) phosphatases constitute a novel class of small, prenylated phosphatases (PRL1, 2, and 3) that share a high degree (>76%) of sequence identity [23–25]. The PRLs are relatively small proteins of about 20 kDa. In addition to the phosphatase domain, there are no regulatory domains except that all PRLs contain a consensus C-terminal prenylation motif CaaX, which is important for their localization to the plasma membrane and early endosomal compartments [23–25]. This family of phosphatases is also known as protein tyrosine phosphatase 4As (PTP4As). PTP4A1/PRL1 was originally identified as an immediate early gene induced during liver regeneration after partial hepatectomy [23]. Subsequently, PTP4A1/PRL1 as well as the closely related PTP4A2/PRL2 and PTP4A3/PRL3 was found to be elevated in numerous tumor cell lines, and cells expressing high levels of PTP4As exhibit enhanced proliferation and anchorage-independent growth [24–29]. Unlike most protein phosphatases that counteract the activity of protein kinases, the PTP4As play a positive role in signaling and possess oncogenic properties [30, 31]. Consistent with their oncogenic potential, we recently revealed that PTP4A2/PRL2 promotes placenta development by downregulating PTEN, leading to AKT activation [32].

Ptp4a2/Prl2 is located on human chromosome 1p35 [30], a region often rearranged or amplified in malignant lymphoma and B-cell chronic lymphocytic leukemia [33, 34]. While Ptp4a2/Prl2 mRNA is highly expressed in pediatric acute myeloid leukemia cells [35, 36], its role in normal and malignant hematopoiesis is largely unknown. Here, we report a functional requirement of PTP4A2/PRL2 in HSC self-renewal. We further demonstrate that PTP4A2/PRL2 is an important mediator of SCF/KIT signaling in HSCs.

Materials and Methods

Mice

The generation of Ptp4a2/Prl2 knockout mice (Ptp4a2/, C57BL6/129P2 mixed background) has been described previously [32]. Ptp4a2/Prl2 knockout mice were backcrossed with C57BL6 mice for at least eight generations. Wild-type C57BL/6 (CD45.2+), B6.SJL (CD45.1+), and F1 mice (CD45.2+ CD45.1+) were purchased from the Jackson Laboratories. All mice were maintained in the Indiana University Animal Facility according to IACUC-approved protocols, and kept in Thorensten units with filtered germ-free air.

Flow Cytometry

Flow cytometry analysis of hematopoietic stem and progenitor cells (HSPCs) was performed as described previously [37, 38]. Murine HSPCs were identified and evaluated by flow cytometry using a single-cell suspension of bone marrow mononuclear cells (BMMCs). Hematopoietic stem and progenitors are purified based upon the expression of surface markers: long-term (LT)-HSC (LinSca1+Kit+CD34CD48CD150+), short-term (ST)-HSC (LinSca1+Kit+CD34+CD48+CD150+), multipotent progenitor cell (MPP) (LinSca1+Kit+CD34+CD48+CD150), common myeloid progenitor (LinSca1IL7RKit+FcγRII/IIIlowCD34high), granulocyte-macrophage progenitor (LinSca1IL7RKit+FcγRII/IIIhighCD34high), megakaryocyte-erythroid progenitor cell (MEP) (LinSca1IL7RKit+FcγRII/IIIlowCD34low), and common lymphoid progenitor (LinIL7R+Sca1lowKitlow). BMMCs were obtained from both tibias and femurs by flushing cells out of the bone using a syringe and Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (FBS). Cells were first stained with a lineage (Lin) cocktail of antibodies from BD Biosciences (biotinylated anti-mouse antibodies directed against CD3e, CD11b, CD45R/B220, Gr-1, and Ter119) as well as Sca-1 PE and c-KIT APC (BD Biosciences, San Jose, CA; http://www.bdbiosciences.com/), and a streptavidin Cychrome conjugate (BD Biosciences, San Jose, CA; http://www.bdbiosciences.com). c-KIT-APC, Sca-1-PE-Cy7, Flt3-PE, CD34-FITC, and streptavidin APC-Cy7 were used for analysis using a FACSLSR II cytometer (BD Biosciences, San Jose, CA; http://www.bdbiosciences.com/). FITC-CD41, FITC-CD48, and FITC-CD34 were purchased from eBioscience and PE-CD150 purchased from Biolegend for SLAM marker analysis. All other antibodies were from BD Biosciences. For immunophenotypic analysis, approximately 3 × 106 BMMCs were stained with antibodies for 30 minutes on ice in dark. Nuclear staining of Ki67 was assessed using an FITC-anti-human Ki67 antibody (BD, Biosciences, San Jose, CA; http://www.bdbiosciences.com/) and fixation and permeabilization solutions from BD Biosciences. Experiments were performed on FACSAria and FACSLSR II cytometers (BD Biosciences, San Jose, CA; http://www.bdbiosciences.com/home.jsp) and analyzed using the FlowJo Version 9.3.3 software (TreeStar, Ashland, OR; http://www.treestar.com/).

BM Transplantation

For the competitive repopulation assays, we injected 5 × 105 BMMCs from wild-type and Ptp4a2 null mice (CD45.2+) plus 5 × 105 competitor BM cells (CD45.1+) into lethally irradiated F1 mice (CD45.1+CD45.2+). Peripheral blood was obtained by tail vein bleeding every month, the RBC lysed, and the Peripheral blood mononuclear cells (PBMCs) stained with anti-CD45.2 FITC and anti-CD45.1 PE, and analyzed by flow cytometry. Sixteen weeks following transplantation, we harvested BM cells from mice reconstituted with wild-type or Ptp4a2 null BM cells and transplanted 3 × 106 BM cells into lethally irradiated F1 mice (CD45.1+CD45.2+).

For HSC transplantation, we injected 200 CD48CD150+KSL cells from wild-type or Ptp4a2 null mice (CD45.2+) plus 3 × 105 competitor BM cells (CD45.1+) into lethally irradiated F1 mice (CD45.1+CD45.2+). Peripheral blood was obtained by retro-orbital eye bleeding every month, the RBC lysed, and the PBMCs stained with anti-CD45.2 FITC and anti-CD45.1 PE, and analyzed by flow cytometry. Sixteen weeks following transplantation, we harvested BM cells from recipient mice and performed flow cytometry analysis to evaluate HSC repopulating capability.

Homing Assays

The homing ability of wild-type and Ptp4a2 null HSPCs was analyzed in irradiated recipient mice. 1 × 107 wild-type or Ptp4a2 null BM cells (CD45.2+) were injected into lethally irradiated recipient (CD45.1+) mice. BM cells were harvested 18 hours following injection and donor-derived cells were evaluated by flow cytometry as CD45.2+ cells, simultaneously identifying Lin-Sca1+CD150+ cells.

Stem and Progenitor Cell Assays

Clonogenic progenitors were determined in methylcellulose medium (MethoCult GF M3234, Stem Cell Technologies, Vancouver, Canada; http://www.stemcell.com/) with cytokines (SCF, TPO, EPO, IL-3, and GM-CSF) using 2 × 104 BMMCs per well (six-well plate). Colonies were scored after 7 days of the initial culture, and all cells were collected and washed twice in phosphate-buffered saline. Subsequently cells were cultured at 2 × 104 per well in the same medium. Colony scoring and replating were repeated every 7 days for at least two times, or until no colonies were observed in the cultures.

Immunoblotting

Cells were washed with ice-cold phosphate-buffered saline, and lysed on ice for 30 minutes in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, and 10% glycerol) supplemented with protease inhibitor (Roche, Basel, Switzerland; http://www.roche.com/) and phosphatase inhibitor (Roche, Basel, Switzerland; http://www.roche.com/). Cell lysates were cleared by centrifugation at 13,200 rpm for 10 minutes, and boiled with Laemmli (SDS)-sample buffer, separated by SDS-PAGE, transferred electrophoretically to a nitrocellulose membrane, and immunoblotted with pAKT (Cell Signaling), AKT (Cell Signaling, Danvers, MA; https://www.cellsignal.com/), pERK1/2 (Cell Signaling, Danvers, MA; https://www.cellsignal.com/), ERK1/2 (Cell Signaling), PTEN (Cell Signaling, Danvers, MA; https://www.cellsignal.com/), PTP4A2/PRL2 (a generous gift from Dr. Qi Zeng), and β-Actin (Santa Cruz, Dallas, Texas; http://www.scbt.com/) followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The blots were developed by the enhanced chemiluminescence technique using the SuperSignal West Pico Chemiluminescent substrate (Fisher Scientific, Hampton, NH; http://www.fishersci.com/) or SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Fisher Scientific, Hampton, NH; http://www.fishersci.com/). Data shown are representation of multiple repeat experiments.

Statistical Analysis

We used either Student's t test or two-way analysis of variance to determine statistical significance. *, p < .05; **, p < .01; ***, p < .001; ns, not significant.

Results

Ptp4a2 Deficiency Results in Inefficient Hematopoiesis

To investigate the role of PTP4A2 in hematopoiesis, we analyzed the peripheral blood and BM of 8–12-week-old wild-type and Ptp4a2/ mice. Ptp4a2/ mice show decreased white blood cell, neutrophil, and lymphocyte counts, whereas hemoglobin levels are normal in the peripheral blood (Supporting Information Fig. S1A). While Ptp4a2 null mice show decreased body size as we previously reported (Supporting Information Fig. S1B) [32], the BM cellularity is normal when normalized to total body weight (Supporting Information Fig. S1C). We also found that Ptp4a2/ mice have smaller spleen and thymus compared with wild-type mice (Supporting Information Fig. S2). These data indicate that PTP4A2 plays an important role in hematopoiesis.

Ptp4a2 Null Mice Have Normal Number of Immunophenotypic HSCs

While Ptp4a1 and Ptp4a2 are broadly expressed in adult tissues including liver [25, 39, 40], the expression pattern of these Ptp4as in the hematopoietic system is unknown. We examined the expression of Ptp4a1, Ptp4a2, and Ptp4a3 during different stages of HSC differentiation using real-time RT-PCR assays. Ptp4a1 and Ptp4a2 are ubiquitously expressed in the hematopoietic compartment, whereas Ptp4a3 expression is most pronounced in MEPs. Ptp4a2 is highly expressed in hematopoietic cells and its expression is enriched in differentiated cells compared to LT-HSCs and committed progenitor cells, suggesting that it could play an important role in lineage commitment (Fig. 1A). We then examined the number of primitive hematopoietic stem/progenitor cells in the BM of Ptp4a2/ mice. Loss of Ptp4a2 slightly increased the frequency (*, p < .05, n = 9; Fig. 1B), but not the absolute number of Kit+Sca1+Lin cells (KSLs) compared to wild-type mice (Fig. 1C); however, Ptp4a2 deficiency neither affects the frequency nor the absolute number of LT-HSCs based on SLAM and CD34 expression (Fig. 1D, 1E). Thus, loss of Ptp4a2 does not affect immunophenotypic HSCs.

Figure 1.

Ptp4a2 null mice have normal number of immunophenotypic hematopoietic stem cells. (A): Real-time RT-PCR analysis of Ptp4a mRNAs in LT-HSCs, ST-HSCs, and representative committed progenitors and differentiated cells. Data shown are the mean values ± SD (n = 3 biological replicates). (B): The frequency of Kit+Sca1+Lin cells (KSLs) was quantified by flow cytometry. Data shown are the mean percentage (±SD) of KSL cells in the bone marrow (*, p < .05, n = 9). (C): The absolute number of KSLs per femur is shown. Graphs represent the mean ± SD (n = 9). (D): The frequency of LT-HSCs (CD34CD48CD150+KSLs) was defined by flow cytometry analysis of SLAM cell surface markers. Data shown are the mean percentage (±SD) of LT-HSCs in the bone marrow (n = 9). (E): The absolute number of LT-HSCs per femur is shown. Graphs represent the mean ± SD (n = 9). Abbreviations: CLPs, common lymphoid progenitors; CMPs, common myeloid progenitors; GMPs, granulocyte-macrophage progenitors; LT-HSCs, long-term hematopoietic stem cells; MEPs, megakaryocyte-erythroid progenitors; MPPs, multi-potential progenitors; ST-HSCs, short-term HSCs.

Ptp4a2 Deficiency Impairs the Long-Term Repopulating Ability of HSCs

To examine whether Ptp4a2 deficiency affects HSC function, we performed serial competitive BM transplantation assays. Equal numbers of donor and competitor BMMCs were transplanted into lethally irradiated recipient mice. Sixteen weeks after primary transplantation, the repopulating ability of Ptp4a2 null cells was significantly lower than wild-type cells (**, p < .01, n = 7; Fig. 2A). Moreover, we found that the percentage of donor-derived T cells was significantly decreased in mice transplanted with Ptp4a2 null cells than that with wild-type cells (11.0% ± 1.9% vs. 4.1% ± 1.7% at 16 weeks, Fig. 2B). Analysis of the BM revealed a striking reduction in the number of phenotypically defined HSCs in the recipients repopulated with Ptp4a2 null BM cells (**, p < .01, n = 5; Fig. 2C). We then transplanted 3 × 106 BMMCs isolated from the primary recipient mice repopulated with wild-type or Ptp4a2 null cells into lethally irradiated secondary recipients. Sixteen weeks after transplantation, Ptp4a2 null cells continued to show decreased repopulating ability (***, p < .001, n = 7; Fig. 2D).

Figure 2.

Ptp4a2 deficiency impairs the long-term repopulating ability of HSCs. (A): Lethally irradiated recipient mice (CD45.1+ and CD45.2+) were transplanted with 5 × 105 bone marrow mononuclear cells (BMMCs) from wild-type and Ptp4a2 null mice (CD45.2+) plus 5 × 105 competitor cells (CD45.1+) in competitive repopulation assays. Graph shows the mean percentage (±SD) of donor-derived (CD45.2+) cells in the peripheral blood post-transplantation, measured at monthly intervals (**, p < .01, n = 7). (B): Contribution of donor-derived cells (CD45.2+) from A, for myeloid cells (Gr1+), B cells (B220+), and T cells (CD3+) in peripheral blood from recipient mice at different time points after transplantation (*, p < .05, n = 7). (C): Quantification of donor-derived (CD45.2+) HSC frequency in the bone marrow of primary recipient mice 18 weeks following transplantation by three phenotypic definitions. Mean ± SD values are shown (**, p < .01, n = 5). (D): Contribution of wild-type and Ptp4a2 null BMMCs to recipient mouse peripheral blood in secondary competitive transplants, measured at monthly intervals (***, p < .001, n = 7). (E): Lethally irradiated recipient mice (CD45.1+ and CD45.2+) were transplanted with 200 LT-HSCs (CD48CD150+KSLs) from wild-type and Ptp4a2 null mice (CD45.2+) plus 3 × 105 competitor cells (CD45.1+) in competitive repopulation assays. Graph shows the mean percentage (±SD) of donor-derived (CD45.2+) cells in the peripheral blood post-transplantation, measured at monthly intervals (***, p < .001, n = 7). Abbreviations: BM, bone marrow; HSC, hematopoietic stem cell; KSL, Kit+Sca1+Lin cell.

To further establish that the self-renewal defect is HSC intrinsic, we purified wild-type and Ptp4a2 null LT-HSCs (CD48-CD150+KSLs) and transplanted 200 of each into lethally irradiated recipient mice along with 300,000 wild-type competitor BMMCs. In this context, Ptp4a2 null HSCs exhibited a substantially lower contribution to peripheral blood production compared to control HSCs (***, p < .001, n = 7; Fig. 2E), demonstrating that Ptp4a2 plays an important role in HSC maintenance and loss of Ptp4a2 impairs HSC self-renewal.

The decreased self-renewal of Ptp4a2 null HSCs in transplantation assays could be due to abnormalities in homing following transplantation. To determine whether Ptp4a2 null BM cells are defective in homing, we performed homing assays and observed similar numbers of donor-derived LinSca-1+Slam+ cells from the BM of recipient mice repopulated with wild-type or Ptp4a2 null BM cells (Supporting Information Fig. S3A), suggesting the absence of homing defects. The impaired HSC self-renewal in Ptp4a2 null mice could also be due to increased apoptosis. Therefore, we evaluated HSC survival by Annexin-V staining and observed no significant difference in the number of apoptotic HSCs (Annexin-V+/PICD48KSLs) in the absence of cytokines (Supporting Information Fig. S3B). Thus, the impaired self-renewal of Ptp4a2 null HSC is likely due to an inability to properly self-renew, rather than homing defects or an increase in apoptosis.

PTP4A2 Promotes HSPC Proliferation

To assess the effect of Ptp4a2 deficiency on proliferation, we stained Ptp4a2 null HSPCs with proliferation marker Ki67. While we detected normal number of Ki67 negative LT-HSCs, we found more Ki67-negative MPPs than normal (**, p < .01, n = 6; Fig. 3A), indicating Ptp4a2 null MPPs are more quiescent and less proliferative. We then performed methylcellulose colony-forming unit (CFU) assays to quantify myeloid progenitor cells. While Ptp4a2 deficiency does not affect the total number of colonies formed, Ptp4a2 null BM cells formed significantly fewer immature GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) colonies compared with wild-type cells (*, p < .05, n = 3; Fig. 3B). Consistent with the cell cycle data, Ptp4a2 null BMMCs show decreased replating potential compared to wild-type cells (**, p < .01, n = 3; Fig. 3C), further demonstrating that hematopoietic progenitor cells show decreased proliferation in the absence of Ptp4a2. To decipher the molecular mechanisms underlying the HSPC proliferation defects seen in the Ptp4a2 null mice, we examined the expression of several known HSC regulators using real-time RT-PCR assays. Several negative regulators of cell cycle, including p21 and p57, are upregulated in Ptp4a2 null HSCs and MPPs, respectively (**, p < .01, n = 3; Fig. 3D, 3E). In addition, the expression of Cyclin D1 (CCND1) and Cyclin E1 (CCNE1) is downregulated in MPPs (**, p < .01, n = 3; Fig. 3E). We further confirmed the changes in p57 and CCND1 expression in MPPs by flow cytometry (Supporting Information Fig. S4).

Figure 3.

PTP4A2 regulates hematopoietic stem and progenitor cell proliferation. (A): Cell-cycle analysis of hematopoietic stem and progenitor cells was performed by staining with DAPI and Ki67 and analyzed by fluorescence-activated cell sorting. Data shown are the mean values ± SD (**, p < .01, n = 6). (B): Myeloid progenitors were quantified by methylcellulose culture using bone marrow mononuclear cells (BMMCs) from wild-type and Ptp4a2 null mice (*, p < .05, n = 3). (C): Serial replating studies. Myeloid progenitors were quantified by methylcellulose culture using BMMCs from wild-type and Ptp4a2 null mice. The methylcellulose cultures were serially replated, weekly, for 3 weeks. Mean values (±SD) are shown (**, p < .01, n = 3). (D, E): Ptp4a2 deficiency results in upregulation of cell cycle regulators in hematopoietic stem cells (HSCs) (D) and MPPs (E). Real-time RT-PCR analysis of some known HSC regulators in LT-HSCs and MPPs. Data shown are the mean values ± SD (**, p < .01, n = 3 biological replicates). Abbreviations: CMPs, common myeloid progenitors; GMPs, granulocyte-macrophage progenitors; KSL, Kit+Sca1+Lin cell; MEPs, megakaryocyte-erythroid progenitors; MPPs, multi-potential progenitors.

The Ability of PTP4A2 to Enhance Hematopoietic Progenitor Cell Proliferation Depends on Its Phosphatase Activity

While we showed that PTP4A2 is important for hematopoietic progenitor cell proliferation, whether this effect depends on its phosphatase activity is not clear. To this end, we overexpressed wild-type or the catalytic inactive mutant form of Ptp4a2 (Ptp4a2/C101S) in Lin cells isolated from wild-type and Ptp4a2 null mice and performed CFU assay [41]. While both wild-type and Ptp4a2 null mononuclear cells form similar number of colonies in CFU assays (Fig. 3B), Ptp4a2 null Lin-Sca1+ cells formed significantly fewer colonies compared with wild-type cells following retroviral transduction (*, p < .05, n = 3; Fig. 4A). Lin-Sca1+ cells are enriched for HSPCs and we showed that Ptp4a2 null progenitor cells are less proliferative (Fig. 3A). Therefore, the difference between the two CFU assays we observed is likely due to distinct composition of cells used in the experiments. Ectopic expression of wild-type or mutant Ptp4a2 did not affect the colony-forming potential of wild-type cells. However, we found that overexpressing wild-type Ptp4a2, but not the mutant form, significantly increase the colony formation of Ptp4a2 null cells (**, p < .01, n = 3; Fig. 4A). We also overexpressed wild-type or the mutant form of Ptp4a2 in Lin cells isolated from wild-type and Ptp4a2 null mice and monitored their proliferation. Consistent with data in the CFU assays (Fig. 4A), mock-transduced Ptp4a2 null Lin cells show decreased proliferation compared with that of mock-transduced wild-type cells (**, p < .01, n = 3; Fig. 4B). Wild-type Ptp4a2 transduced Ptp4a2 null cells showed enhanced proliferation compared with mock and mutant Ptp4a2 transduced cells (**, p < .01, n = 3; Fig. 4B).

Figure 4.

The ability of PTP4A2 to enhance hematopoietic progenitor cell proliferation depends on its phosphatase activity. (A): Myeloid progenitors were quantified by methylcellulose culture using wild-type and Ptp4a2 null Lin− cells transduced with retroviruses expressing the WT or the mutant form (Ptp4a2/C101S) of Ptp4a2. Data are means ± SD (**, p < .01, n = 3 independent experiments). (B): Proliferation of wild-type and Ptp4a2 null Lin cells expressing the WT or the mutant form (Ptp4a2/C101S) of Ptp4a2 in response to SCF stimulation. Data are means ± SD (**, p < .01, n = 3 independent experiments). (C): Immunoblot analysis of AKT and ERK phosphorylation in freshly purified wild-type and Ptp4a2 null Lin cells. *, p < .05 compared with wild-type cells by one-way analysis of variance (ANOVA) and Bonferroni post hoc test, n = 3 independent experiments. Representative Western blot analysis of indicated proteins is shown. (D): Immunoblot analysis of AKT and ERK phosphorylation in Ptp4a2 null Lin cells expressing the WT or the mutant form (Ptp4a2/C101S) of Ptp4a2. *, p < .05 compared with mock transduced cells by one-way ANOVA and Bonferroni post hoc test, n = 3 independent experiments. Representative Western blot analysis of indicated proteins is shown.

To decipher the molecular mechanisms underlying the HSPC proliferation defects seen in the Ptp4a2 null mice, we examined the activation of AKT and ERK signaling in freshly purified Lin cells. We observed that the levels of pAKT and pERK1/2 are significantly lower in Ptp4a2 null Lin− cells compared with those in wild-type cells (Fig. 4C). To investigate the mechanism by which Ptp4a2 deficiency attenuates AKT and ERK activation, we analyzed PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression in both wild-type and Ptp4a2 null cells. PTEN is an antagonist of PI3K signaling pathway [42]. In addition, PTEN has also been shown to negatively regulate the activation of the ERK pathway [42]. We previously reported that in placenta, Ptp4a2 deficiency led to elevated PTEN expression [32]. To examine whether this is also the case in HSPCs, we measured PTEN protein level in wild-type and Ptp4a2 null Lin− cells. As shown in Figure 4C, PTEN expression was 1.4 ± 0.15-fold higher in Ptp4a2 null samples, consistent with our previous observation in placenta [32]. Therefore, impaired AKT and ERK activation in Ptp4a2 null Lin− cells are likely due to increased PTEN level. Furthermore, we found that expression of wild-type Ptp4a2, but not the catalytically inactive mutant Ptp4a2, significantly augments pAKT and pERK1/2 levels in Ptp4a2 null Lin cells (Fig. 4D and Supporting Information Fig. S5), demonstrating that the ability of PTP4A2 to promote hematopoietic progenitor cell proliferation and activation of AKT and ERK signaling depends on its phosphatase activity.

PTP4A2 Is Important for SCF/KIT Signaling in HSPCs

Given that Ptp4a2 null BMMCs show decreased colony formation in serial replating assays, PTP4A2 may play an important role in cytokine signaling. To test this hypothesis, we cultured both wild-type and Ptp4a2 null Kit+ cells in the presence or absence of cytokines, including SCF, G-CSF, and IL-3, and monitored their proliferation. While wild-type and Ptp4a2 null Kit+ cells exhibited similar proliferative behaviors in response to G-CSF or IL-3 stimulation, Ptp4a2 null hematopoietic progenitor cells showed decreased proliferation in response to SCF (*, p < .05, n = 3; Fig. 5A). In CFU assays, Ptp4a2 null KSL cells formed significantly fewer colonies compared with wild-type KSL cells in response to SCF stimulation (**, p < .01, n = 3; Fig. 5B). Importantly, Ptp4a2 null KSL cells showed marked reduction of GEMM colony formation in response to decreased concentration of SCF compared with wild-type KSL cells, demonstrating that PTP4A2 is essential for the proliferation of hematopoietic progenitor cells following SCF stimulation. We then examined whether Ptp4a2 deficiency affects HSPC survival in the absence of cytokine. As shown in Figure 5C, Ptp4a2 null Kit+ cells showed decreased cell counts compared with wild-type cells (*, p < .05, n = 3), indicating that PTP4A2 is essential for HSPC survival in the absence of cytokine. Furthermore, we observed that the levels of pAKT and pERK1/2 are also significantly lower in Ptp4a2 null Kit+ cells compared with those in wild-type cells in the basal state (Fig. 5D). Stimulation of HSPCs with SCF can activate several signaling pathways through KIT, including PI3K/AKT and ERK1/2 pathways [4–6]; therefore, it is possible that Ptp4a2 deficiency impairs the activation of these signaling pathways in response to SCF stimulation. This is indeed the case, as Ptp4a2 null cells displayed decreased activation of both pAKT and pERK1/2 at 30 minutes following SCF stimulation (Fig. 5D), demonstrating that PTP4A2 is important for sustaining SCF signaling in HSPCs.

Figure 5.

PTP4A2 is important for SCF/KIT signaling in hematopoietic stem and progenitor cells. (A): Proliferation of wild-type and Ptp4a2 null Kit+ cells in response to cytokine stimulation. Data shown are the mean values ± SD (*, p < .05, n = 3). (B): Colony-forming unit assays in the presence of increased concentration of SCF. Data shown are the mean values ± SD (**, p < .01, n = 3). (C): Proliferation of wild-type and Ptp4a2 null Kit+ cells in the absence of cytokines. Data shown are the mean values ± SD (*, p < .05, n = 3). (D): Immunoblot analysis of AKT and ERK phosphorylation in wild-type and Ptp4a2 null Kit+ cells. Data represent three independent experiments. Abbreviation: SCF, stem cell factor.

PTP4A2 Is Important for Oncogenic KIT Signaling in Hematopoietic Progenitor Cells

Hematopoietic cell lines and primary BM cells expressing the oncogenic KIT/D814V mutant display ligand-independent proliferation in vitro and MPD in vivo [18–22]. Based on our finding that PTP4A2 is important for SCF/KIT signaling in HSPCs, we speculated that PTP4A2 may also enhance KIT/D814V-mediated ligand-independent hyperproliferation and activation of signaling pathways. To test this, we introduced wild-type KIT and KIT/D814V mutant into Lin cells purified from wild-type and Ptp4a2 null mice and performed serial replating assays. In primary (*, p < .05, n = 3), secondary (**, p < .01, n = 3), and tertiary replating experiments (***, p < .001, n = 3), Ptp4a2 null Lin cells expressing KIT/D814V formed significantly fewer colonies compared with wild-type Lin cells expressing the KIT mutant (Fig. 6A). Both wild-type and Ptp4a2 null cells expressing wild-type KIT did not form any colonies in the tertiary replating experiments (Fig. 6A). To determine whether PTP4A2 is essential for KIT/D814V-mediated ligand-independent growth, we cultured Ptp4a2 wild-type and null cells expressing wild-type or KIT/D814V in the absence of cytokine or the presence of SCF. As shown in Figure 6B (**, p < .01, n = 3), Ptp4a2 deficiency significantly decreased the ability of KIT/D814V to promote cell proliferation both in the absence and presence of SCF. Furthermore, we observed decreased phosphorylation of AKT and ERK1/2 in Ptp4a2 null cells expressing KIT/D814V mutant compared with wild-type cells (Fig. 6C). Thus, PTP4A2 is essential for KIT/D814V-mediated hematopoietic progenitor cell hyperproliferation and activation of signaling pathways.

Figure 6.

PTP4A2 is important for oncogenic KIT signaling in hematopoietic progenitor cells. (A): Myeloid progenitors were quantified by methylcellulose culture in the presence of SCF using wild-type and Ptp4a2 null Lin cells transduced with retroviruses expressing the WT or the mutant form (KIT/D814V) of KIT. The methylcellulose cultures were serially replated, weekly, for 3 weeks. Data are means ± SD (*, p < .05; **, p < .01; ***, p < .001, n = 3 independent experiments). (B): Proliferation of wild-type and Ptp4a2 null Lin cells expressing the WT or the mutant form (KIT/D814V) of KIT in the presence or the absence of cytokine stimulation. Data are means ± SD (**, p < .01, n = 3 independent experiments). (C): Immunoblot analysis of AKT and ERK phosphorylation in wild-type and Ptp4a2 null Lin cells expressing the WT or the mutant form (KIT/D814V) of KIT in response to SCF stimulation. Data represent three independent experiments. Abbreviation: SCF, stem cell factor.

Discussion

The PTP4A family of phosphatases, consisting of PTP4A1, PTP4A2, and PTP4A3, represents an intriguing group of proteins implicated as biomarkers and therapeutic targets in cancer [23–25]. Individual PTP4As are overexpressed in a variety of cancer cell lines and tissues when compared with their normal counterparts [26–29]. However, the exact biological functions of PTP4As are largely unknown [23–25]. Herein we report that PTP4A2/PRL2 plays a critical role in HSC self-renewal and SCF/KIT signaling.

While loss of Ptp4a2/Prl2 modestly increased the frequency of MPPs, Ptp4a2 deficiency does not affect immunophenotypic HSC numbers. To further define the role of PTP4A2 in regulating HSC self-renewal, we performed serial bone marrow transplantation assays and found that loss of Ptp4a2 impairs the ability of HSCs to repopulate the lethally irradiated recipient mice. These observations were confirmed by HSC transplantation. Furthermore, we observed no homing defects following transplantation and no increased HSC apoptosis, demonstrating that PTP4A2 functions as a positive regulator of HSC self-renewal. We also found that the percentage of donor-derived T cells was significantly lower in mice transplanted with Ptp4a2 null cells than that with wild-type cells, indicating that PTP4A2 may also regulate T-cell differentiation under stress conditions.

Among the signal transduction pathways that have attracted considerable attention as possibly being involved in HSC maintenance is the phosphoinositide 3-kinase (PI3K)-AKT pathway [43]. As an antagonist of the PI3K pathway, PTEN has been implicated as a regulator of HSC self-renewal and loss of PTEN in the HSC compartment results in hyperproliferation of HSPCs [44]. Recently, we showed that in mouse placenta PTP4A2/PRL2 promotes cell proliferation by activating the AKT kinase through downregulation of the tumor suppressor PTEN [32]. It appears that Ptp4a2 overexpression enhances the degradation of PTEN protein in these cells, indicating that PTP4A2/PRL2 regulates the stability of PTEN in placenta [32]. In addition, we observed that loss of Ptp4a2 decreases HSPC proliferation and Ptp4a2 null hematopoietic progenitor cells show decreased phosphorylation of AKT and ERK1/2 in steady state. To investigate how PTP4A2 regulates PI3K/AKT signaling in hematopoietic progenitor cells, we examined the level of PTEN in Ptp4a2 null HSPCs. As seen in placenta, we observed moderate but statistically significant elevation of PTEN protein in Ptp4a2 null HSPCs compared to that in wild-type HSPCs, whereas PTEN RNA level is identical between Ptp4a2 wild-type and null cells. Therefore, it is possible that PTP4A2 regulates the stability and turnover of PTEN protein in HSPCs as well. PTEN has been shown to regulate cell proliferation and cancer progression in a dosage-dependent manner and a slight reduction in PTEN levels dictates cancer susceptibility [45, 46]. Thus, it is likely that increased level of PTEN we observed contributes to the decreased activation of AKT and ERK1/2 signaling pathways in Ptp4a2 null HSPCs. Moreover, consistent with PTEN's established role in sustaining HSPC proliferation, our data suggest that increased PTEN expression in Ptp4a2 deficient mice leads to reduced proliferation of HSPCs.

Ectopic expression of Ptp4a2 in nontumorigenic cells has been shown to enhance cell cycle progression and promotes proliferation [28, 36]. This process may be dependent on the downregulation of cyclin-dependent kinase inhibitor p21 [28]. The cell cycle defects seen in Ptp4a2 null mice may also be mediated by cell cycle regulators as we observed increased expression of p21 and p57 in HSPCs. Both p21 [47] and p57 [48] have been shown to regulate HSPC proliferation; therefore, PTP4A2 plays an important role in promoting HSPC proliferation, at least in part by controlling the level of cell cycle regulators. How PTP4A2 regulates the expression of these cell cycle regulators is not clear. However, we note that PTEN controls cell cycle entry and progression through inhibiting PI3K-AKT activity [49, 50] and PTEN loss leads to enhanced G0-G1 cell cycle transition, which yields a ST expansion of HSCs [43].

Given that Ptp4a2 null hematopoietic progenitor cells show decreased colony formation in serial replating assays, it is possible that PTP4A2 regulates cytokine signaling in hematopoietic progenitor cells. While Ptp4a2 null hematopoietic progenitor cells proliferate normally in response to IL-3 or G-CSF stimulation, these cells are less proliferative in response to SCF stimulation. Furthermore, Ptp4a2 null hematopoietic progenitor cells show decreased colony formation in response to increased concentrations of SCF compared with wild-type cells. The role of PTP4A2 in mediating SCF/KIT signaling appears modest, as SCF still activates a remarkable amount of AKT and ERK in Ptp4a2 null cells (Fig. 5D). Therefore, PTP4A2 may be one of many factors that contribute to KIT function in HSPCs.

As the receptor for SCF, the biologic significance of KIT in hematopoiesis was first revealed in the white spotting (W) mutant mice [51]. Several W mutations with different levels of KIT kinase deficiency were found to correlate with the phenotype severity [9–14]. Studies in the viable primary W mutant mice or in mice after transplantation indicate the importance of KIT signaling in maintaining HSC quiescence and survival [9–14]. The hematopoietic defects seen in the Ptp4a2 null mice recapitulate some hematopoietic phenotype of KIT mutant mice, providing additional evidence that PTP4A2 plays an important role in mediating SCF/KIT signaling in hematopoietic progenitor cells.

While Ptp4a2 is highly expressed in acute myeloid leukemia cells [35, 36], its role in leukemogenesis is largely unknown. We discovered that loss of Ptp4a2 decreased the ability of KIT/D814V mutant in promoting hematopoietic progenitor cell proliferation, demonstrating that PTP4A2 is an important effector molecule of oncogenic KIT signaling in HSPCs and positively contributes to KIT/D814V-induced hematopoietic progenitor hyperproliferation. Therefore, pharmacological inhibition of PTP4A2 in KIT/D814V-expressing cells in vivo could delay leukemia progression and decrease the severity of the disease.

Conclusions

In summary, this study reveals a critical role of PTP4A2/PRL2 in HSC self-renewal and SCF/KIT signaling. Our results suggest that the PTP4A2 phosphatase may be a druggable target in MPD and AML with oncogenic KIT mutations.

Acknowledgments

This work was supported in part by National Institutes of Health Grant CA69202 (Z.-Y.Z.), Department of Defense Grant CA120373 (Y.L.), an Elsa Pardee Foundation Research Grant (Y.L.), an American Cancer Society Institutional Research Grant (Y.L.), an Indiana University Simon Cancer Center Pilot Project Grant (Y.L.), and an Indiana University Biomedical Research Grant (Y.L.). We would like to thank Marilyn Wales for helping in the preparation of the manuscript.

Author Contributions

M.K.: designed the research, performed the research, analyzed the data, performed the statistical analysis, and wrote the manuscript; R.K., M.C.Y., and Z.-Y.Z.: designed the research and wrote the manuscript; Y.L.: designed the research, analyzed the data, performed the statistical analysis, and wrote the manuscript; Y.D. and H.Y.: performed the research, analyzed the data, and performed the statistical analysis; Y.B., S.C., and L.Z.: performed the research.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

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