Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling

Bone marrow contains types of stem cell that can produce new blood and immune cells. Transplanting bone marrow from a healthy person can be used to treat people with certain disorders of the blood and immune system, by providing a new supply of regenerating bone marrow stem cells. Bone marrow transplants are also critical for individuals who had their own bone marrow stem cells destroyed by cancer or by toxic anti-cancer therapies like chemotherapy.

Acquiring bone marrow for transplants can be a difficult process. Sometimes doctors can use the patient's own stem cells for a transplant. But in circumstances where the patient lacks healthy bone marrow, a bone marrow donor must be found. Donors and recipients must be carefully matched: certain proteins on the donor's bone marrow cells must be very similar to the proteins on the recipient's bone marrow cells, or the recipient's immune system will attack and kill the new cells.

If scientists could learn to grow the stem cells found in bone marrow in laboratories, they could circumvent some of the challenges associated with bone marrow donation. To do that, scientists must first understand the precise molecular mechanisms that allow the blood cell-producing stem cells to regenerate themselves and produce new blood cells.

Angiopoietin-like proteins are commonly used to help stem cells grow in the laboratory, and Lin et al. have now looked in detail at how these proteins work. This revealed that angiopoietin-like proteins also cause the stem cells that produce blood cells to grow in zebrafish embryos and are necessary for the embryos' vascular system to develop properly.

Zebrafish embryos lacking angiopoietin-like proteins develop a similar set of developmental problems as zebrafish embryos with a mutation in another protein called Notch. Through a series of experiments, Lin et al. show that angiopoietin-like proteins interact with Notch and help transform Notch into its active form, which is necessary for blood stem cell growth. Lin et al. also found that both angiopoietin-like proteins and Notch affect the same signaling molecules. This suggests that the two proteins may work together as part of the same molecular pathway. The work suggests an alternative method to activate Notch for blood stem cell stimulation during processes such as bone marrow or cord blood transplantation.

Human hematopoietic stem and progenitor cells (HSPCs) are defined as cells with the ability to self-renew and differentiate into all blood lineages. They provide tremendous therapeutic potential for bone marrow transplantation in the treatment of hematologic malignancy, inherited blood disorders, and cancer chemotherapy. An important goal in studying stem cells is to identify factors that can expand HSPCs in vitro, while maintaining their self-renewal capacity. Scientists have turned to clues during embryonic hematopoietic development to identify novel factors that regulate this expansion. Developmental hematopoiesis occurs in at least two distinct phases, in which an initial transient wave produces mainly primitive erythrocytes and myeloid cells, followed by a definitive wave which produces long-term HSPCs (Orkin and Zon, 2008). Ontogeny studies across species have identified the aorta-gonad-mesonephros (AGM) region as the site from which definitive HSPCs arise (Dieterlen-Lievre, 1975; Medvinsky and Dzierzak, 1996; Tavian et al., 1996; Jaffredo et al., 1998). Recent studies using time-lapse imaging in live zebrafish embryos and live mouse thick tissue sections revealed that HSPCs bud off from the endothelium lining the ventral wall of the developing dorsal aorta (DA) prior to entering circulation (Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010). Subsequent to their birth in the AGM, HSPCs migrate to the fetal liver where they undergo significant expansion in vivo (Ema and Nakauchi, 2000) before colonizing the bone marrow and provide life-long supply of all blood cells.

Angiopoietin-like proteins (ANGPTLs) were recently identified as growth factors capable of expanding mouse (Zhang et al., 2006; Zheng et al., 2011; Farahbakhshian et al., 2014) and human (Zhang et al., 2008; Khoury et al., 2011; Ventura Ferreira et al., 2013; Fan et al., 2014) HSPCs in culture. ANGPTLs are secreted proteins that closely resembled the Angiopoietins, which are important vascular regulators, but despite structural similarities, they do not bind to TIE-2 or TIE-1 (Kim et al., 1999, 2000; Oike et al., 2004). They also exert much wider functions outside of the vasculature such as regulation of lipid, glucose and energy metabolism (Hato et al., 2008), inflammation (Tabata et al., 2009), and cancer (Zhu et al., 2011).

Notch receptors (NOTCH1-4) are single-pass type I transmembrane receptors implicated in various developmental and disease processes including the activation of the hematopoietic program. They are synthesized as ∼300 kD full-length precursor proteins that undergo a series of proteolytic cleavages in order to become fully activated. Prior to translocating to the cell surface, NOTCH is first cleaved by furin-like convertases in the trans-Golgi compartment, resulting in a heterodimer composed of the N-terminal extracellular domain and the C-terminal-transmembrane/intracellular domain, bound through noncovalent linkage. Canonical NOTCH activation requires its obligatory interaction with NOTCH ligands belonging to the Delta/Serrate/Jagged/LAG-2 (DSL) family (Fortini, 2009; Kopan and Ilagan, 2009). Several studies support a model in which ligand binding followed by endocytosis creates a mechanical force that alters the conformation of a juxtamembrane NOTCH negative regulatory region (Kopan and Ilagan, 2009). This permits cleavage of NOTCH by ADAM/TACE (a disintegrin and metalloprotease/tumor necrosis factor α converting enzyme) at Site 2 (S2), generating the membrane-anchored NOTCH extracellular truncation (NEXT) fragment. NEXT is a substrate for the γ-secretase complex, which cleaves Site 3 (S3), releasing the NOTCH intracellular domain (NICD) to freely translocate into the nucleus to interact with DNA binding protein CSL (CBF1/Su(H)/Lag-1)/RBPjκ and initiate transcription of target genes.

While the role of NOTCH in adult HSPC homeostasis still remains controversial, NOTCH is irrefutably important during embryonic hematopoiesis. Several Notch receptors and ligands are expressed in the mouse AGM and deletion of Notch1, Jagged1, or CSL resulted in impaired intra-embryonic hematopoiesis (Kumano et al., 2003; Robert-Moreno et al., 2005, 2008). Notch target genes such as Gata2 (Minegishi et al., 2003), Runx1 (North et al., 2002) and those belonging to the Hairy and Enhancer-of-split related basic helix-loop-helix transcription factors, Hes1, Hey1, and Hey2, are also expressed in the AGM (Robert-Moreno et al., 2005, 2008; Guiu et al., 2013). Previous studies from our lab demonstrated that HSPC fate is dictated by the notch-runx1 pathway, in which overexpression of runx1 mRNA in the notch mutant mindbomb (mib) can partially restore the loss of HSPCs normally observed in mib (Burns et al., 2005). Furthermore, recent studies demonstrated an even earlier role for notch in which somite-derived signals such as wnt16 (Clements et al., 2011) or physical intracellular contacts between the jam1a/2a adhesion proteins (Kobayashi et al., 2014) can regulate notch signaling in HSC precursors.

Because of their potential in hematological applications and therapy, it is important to decipher the molecular pathways on which these ANGPTLs act. Here, we utilized zebrafish genetics to help provide insights into the mechanism by which ANGPTLs can expand adult HSPCs. We found that angptls1 and 2 are indispensible for zebrafish definitive hematopoiesis and that they genetically interacted with notch signaling. To further uncover potential mechanisms for this interaction, we utilized cultured human cells and found that ANGPTL2 mediates NOTCH receptor cleavage/activation, occurring at the level of ANGPTL receptor binding to NOTCH. Our novel findings that angptls can induce notch activation provide an additional layer of regulation of canonical notch signaling.

Previous studies have demonstrated that notch is indispensable during the developmental specification of HSPCs (Burns et al., 2005; Clements et al., 2011). In our present study, we defined the essential role of zangptl1 and 2 during developmental hematopoiesis (Figure 1), likely through regulation of notch signaling in the hemogenic endothelium. To our knowledge, this is the first time zangptls has been shown to activate notch. The current model for notch specification of HSPCs during development has mostly been suggested to be a cell-autonomous one (Hadland et al., 2004; Burns et al., 2005; Robert-Moreno et al., 2005). Our live imaging data from transient Tg(drl:eGFP;kdrl:NICD)-injected embryos resulting in significantly more budding HSPCs in the AGM of DA corroborate with this. More recently, studies have revealed an additional and separate role for somite-derived notch signaling during HSPC specification that occurs much earlier than previously thought (Clements et al., 2011; Kobayashi et al., 2014). Based on our genetic data in zebrafish in which we observed a tight genetic interaction between zangptls and notch (Figure 2), we believe that zangptls exert their effects on definitive hematopoiesis during the vascular specifications of the axial vessels including the DA, when it coincides with the highest endogenous expression of zangptl2 (Figure 1—figure supplement 1B). Though it remains to be determined, information on the spatial/temporal expression pattern of the yet unidentified zangptl receptor would solidify this premise. There are receptors in zebrafish with similar domains as the mammalian receptor, but there is no obvious orthologue. Nevertheless, our current proposed model (Figure 7A) extrapolated from our genetic data places zangptl2 downstream of mib as Tg(hsp70:zangptl2) crossed into mib can rescue its hematopoietic defect (Figure 2C). Zangptl2 acts upstream of notch activation, that is, generation of NICD, as forced expression of NICD from Tg(hsp70:Gal4;UAS:NICD) can rescue angptls-MO morphant phenotype (Figure 2B). Interestingly, loss of notch signaling in angptls-MO-injected notch reporter embryos (Figure 2A) indicates that zangptls are required for notch signaling but also raises questions as to whether notch ligands needed to be present for zangptls to regulate notch activation. One of the notch ligands, deltaC, has considerable overlapping expression both spatially and temporally with notch in the developing DA, suggesting that it may play a role during the formation of the hemogenic endothelium and the subsequent generation of HSPCs. DeltaC expression, normally absent in the DA of mib was similarly rescued in Tg(hsp70:zangptl2);mib (data not shown). This restoration of notch ligand in the mib by zangptl2 overexpression was intriguing, and it remains to be established whether this restored notch ligand is functional in the absence of mib.

Figure 7

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To examine potential downstream signaling from angptls and notch, we looked at the previously identified target of notch, myc. The relationship between NOTCH and MYC has been best studied in T-ALL cells whereby MYC transduces growth and survival signals to many NOTCH-dependent T-ALL cell lines and forced ectopic expression of MYC can restore the leukemogenic signals for these cells in instances when NOTCH is inhibited (Palomero et al., 2006; Weng et al., 2006; Chan et al., 2007). However, myc has not been studied in zebrafish hematopoiesis. We found that not only is myc required during HSPC formation in the AGM (Figure 4A), overexpression of exogenous myc in embryos lacking zangptls (angptls-MO) or notch (mib) signaling could similarly rescue AGM defects (Figure 4C,B). This puts myc downstream of zangptls and notch signaling. Through independent gene expression analysis, we found MYC to be highly activated upon ANGPTL2 stimulation in human CD34⁺ progenitor cells (Figure 4D). Furthermore, we ran GSEA comparing our ANGPTL2-induced gene expression signature to the set of MYC target genes whose regulatory elements were bound by NOTCH from our ChIP-seq data and found significant overlap (Figure 4E). This implies that the set of MYC targets downstream of ANGPTL2 and NOTCH signaling is highly similar, supporting our hypothesis that ANGPTL may signal partially through NOTCH.

To test this hypothesis, we utilized cultured mammalian cells to directly investigate the mechanism by which ANGPTL2 may act on NOTCH. We first discovered that purified ANGPTL2 ligand added to cells can rapidly induce NOTCH activation, measurable using three different assays: a luciferase reporter assay (Figure 5—figure supplement 1A), qPCR of known NOTCH target genes (Figure 5B and Figure 5—figure supplement 1B), and Western blotting for the NOTCH cleavage product NICD (Figure 5A). Of particular note, when we used different drugs that block certain cleavage events, we found that ANGPTL2-stimulated S2 cleavage of NOTCH (Figure 5C). Interestingly, this step occurs at the cell surface when NOTCH engages with its ligands. Although the precise mechanism in which ANGPTL2 mediates NOTCH cleavage is subject to further investigation, our assumption that this level of regulation must occur in close proximity led to our surprising discovery that human ANGPTL2 receptor, LILRB2, can interact with NOTCH (Figure 6A–D and Figure 6—figure supplement 1,2). These preliminary results from co-IPs certainly suggest that ANGPTL signaling occurs in very close proximity to NOTCH. Such co-IP experiments do not definitively signify that there is direct physical interaction and do not address the stoichiometry of this potential interaction. Nevertheless, ANGPTL2-mediated NOTCH cleavage appears to be dependent on LILRB2 signaling, as sh-LILRB2 knockdown resulted in diminished NICD generation (Figure 6E). This demonstrates a functional interaction. A more thorough mapping of the interactions between these two surface receptors will shed light on the mechanism by which ANGPTL2 can regulate NOTCH activation. An attractive speculation as to why this interaction may occur would be to potentially facilitate or even poise the NOTCH receptor for more efficient cleavage, as depicted in the model proposed in Figure 7B. This also raises an intriguing possibility of whether LILRB2 can physically alter NOTCH interaction with its ligand or whether ANGPTL2 signaling can influence NOTCH ligand availability. Indeed several studies have suggested that Notch receptors oligomerize at the cell surface at sites of DSL ligand contact from neighboring cells (Luty et al., 2007; Nichols et al., 2007). The recruitment of LILRB2 to NOTCH may facilitate this clustering process although the stoichiometry of LILRB2 to NOTCH remains to be established.

Various alternative non-DSL type ligands have been identified to activate NOTCH, such as the adhesion molecule F3/Contactin (Hu et al., 2003), EGF-repeat factor DNER (Eiraku et al., 2005), EGF-like domain 7 (Schmidt et al., 2009), and Microfibrillar associated glycoproteins (MAGP) 1 and 2 (Miyamoto et al., 2006). Interestingly, a related family member to the latter, MFAP4 was found to be capable of expanding mouse HSPCs ex vivo along with ANGPTLs 2 and 3 (Zhang et al., 2006). Our study does not propose ANGPTL2/LILRB2 as a non-canonical NOTCH ligand but offers an additional layer of regulation on NOTCH activation which may not be previously appreciated.

Previous report on the discovery of ANGPTL receptor, LILRB2, suggested downstream activation of CAMK (calcium/calmodulin-dependent protein kinase)-2 and -4 and subsequent recruitment of SHP-2 (a Src homology 2 domain containing non-transmembrane protein tyrosine phosphatase) in freshly isolated mouse spleen cells (Zheng et al., 2012). CAMK2 has been shown to activate NOTCH in human prostate cancer cells (Mamaeva et al., 2009) whereas SHP-2 has also been shown to genetically interact with notch signaling in Drosophila (Oishi et al., 2006). Although the answer as to whether CAMKs and SHP-2 may directly or indirectly regulate NOTCH is still yet to be determined, this suggests that LILRB2 signaling may extend beyond the models presented in our studies.

The ability of ANGPTLs to regulate the local events at which NOTCH cleavage occurs creates new opportunity for therapeutic intervention. The role of Notch during HSPC renewal at homeostasis is controversial (Bigas et al., 2010). Recent studies demonstrate that endothelial-derived Notch signal can stimulate murine cKit⁺Sca1⁺Lin⁻ cells expansion (Butler et al., 2010). Delta1^ext−IgG, in which the engineered activating NOTCH ligand is tethered to the Fc domain of human IgG1, was developed for ex vivo expansion of human CD34⁺ cord blood (CB) progenitors (Delaney et al., 2005). More recently, Delta1^ext−IgG-expanded HSPCs showed great clinical promise in rapid hematopoietic engraftment with shortened time to neutrophil recovery (Delaney et al., 2010). In support of an interaction between the angptls and notch, preliminary analysis of human CD34⁺ CB progenitors treated with ANGPTL2 or Delta^ext−IgG had comparable expansion in CD34⁺/CD90(Thy-1)^lo populations. Furthermore, these expansions were effectively abrogated when cells were treated with a combination of NOTCH1 and NOTCH2 blocking antibodies, suggesting that they were at least partially NOTCH mediated. This supports that the ANGPTLs and NOTCH can interact in another cell type such as human cord blood CD34⁺ cells. In conclusion, our studies of ANGPTL-mediated activation of NOTCH presented here have uncovered an important genetic, physical, and functional interaction of these two signaling pathways that are critical for hematopoiesis.

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Author details

1. Michelle I Lin

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   MIL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Emily N Price

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   ENP, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

3. Sonja Boatman

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   SB, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

4. Elliott J Hagedorn

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   EJH, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

5. Eirini Trompouki

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   ET, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

6. Sruthi Satishchandran

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   SS, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

7. Charles W Carspecken

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   CWC, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

8. Audrey Uong

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   AU, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

9. Anthony DiBiase

   Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   ADB, Bioinformatics analysis

   Competing interests

   No competing interests declared.

10. Song Yang

    Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

    Contribution

    SY, Bioinformatics analysis

    Competing interests

    No competing interests declared.

11. Matthew C Canver

    Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

    Contribution

    MCC, Acquisition of data, Contributed unpublished essential data or reagents

    Competing interests

    No competing interests declared.

12. Ann Dahlberg

    Pediatric Oncology, Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, United States

    Contribution

    AD, Acquisition of data, Analysis and interpretation of data

    Competing interests

    No competing interests declared.

13. Zhigang Lu

    1. Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, United States

    Contribution

    ZL, Provided reagents and useful information

    Competing interests

    No competing interests declared.

14. Cheng Cheng Zhang

    1. Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Physiology and Developmental Biology, University of Texas Southwestern Medical Center, Dallas, United States

    Contribution

    CCZ, Provided reagents and useful information, Drafting or revising the article

    Competing interests

    No competing interests declared.

15. Stuart H Orkin

    1. Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States
    2. Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, United States

    Contribution

    SHO, Drafting or revising the article, Contributed unpublished essential data or reagents

    Competing interests

    No competing interests declared.

16. Irwin D Bernstein

    Pediatric Oncology, Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, United States

    Contribution

    IDB, Drafting or revising the article, Contributed unpublished essential data or reagents

    Competing interests

    No competing interests declared.

17. Jon C Aster

    Department of Pathology, Brigham and Women's Hospital, Boston, United States

    Contribution

    JCA, Provided reagents and useful information, Conception and design, Drafting or revising the article

    Competing interests

    No competing interests declared.

18. Richard M White

    1. Department of Cancer Biology, Memorial Sloan Kettering Cancer Center, New York, United States
    2. Department of Genetics, Memorial Sloan Kettering Cancer Center, New York, United States
    3. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, United States

    Contribution

    RMW, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    No competing interests declared.

19. Leonard I Zon

    Stem Cell Program and Division of Hematology/Oncology, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, United States

    Contribution

    LIZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    zon@enders.tch.harvard.edu

    Competing interests

    LIZ: I am a founder and stockholder of Fate, Inc. and Scholar Rock, and a scientific advisor for Stemgent.

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