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X. Wang et al.
vate mTORC1.5,6 Rheb binds to mTORC1 at the lyso- some and activates it, while TSC1-TSC2-TBC1D7, a GTPase-activating protein, inhibits mTORC1 activity by reducing GTP-bound Rheb.7-9 mTORC1 is a serine/threo- nine (Ser/Thr)-protein kinase complex that responds to multiple signals, regulates cell biological activities, and maintains homeostasis.10 Dysregulation of the mTORC1 signaling pathway results in myeloproliferative neoplasms (MPN) and, in some cases, acute leukemia, making mTORC an important target for therapeutic treatments.10 Perturbation of mTORC1 in adult mouse HSCs by Raptor deletion results in the depletion of the long-term reconsti- tution ability of HSCs.11 Deletion of TSC1, an upstream negative regulator of mTORC1, causes defective HSC cycling and adversely affects HSC function in mice due to enhanced mTORC1 activity,12 indicating that the level of mTORC1 activity needs to be precisely regulated. Changes in mTORC1 activity in the hematopoietic sys- tem under various conditions alters HSC function and homeostasis.
Rheb1 can also regulate cell proliferation and apoptosis via interaction with many other signaling pathways, such as B-Raf, Notch, small Rho GTPase and Akt signaling pathways. Knockdown of Rheb1 in a TSC2-null, angiomyolipoma-derived cell line decreased Notch activi- ty, suggesting that Notch is a downstream target of Rheb1. However, Notch activation could not be blocked by rapamycin, the mTORC1 inhibitor.13 Rheb1 was also reported to directly interact with the B-Raf kinase in a rapamycin-resistant manner and inhibit its function, resulting in interference with H-Ras-induced transforma- tion in NIH3T3 cells.14 In addition, Rheb1 interacts with FKBP38 and regulates apoptosis in a rapamycin-insensi- tive, but amino acid- and serum-sensitive manner.15 We have previously reported that Rheb1 plays a crucial role in myeloid development. The expression of Rheb1 is high in myeloid progenitor, and is down-regulated during granu- locyte differentiation. Rheb1 deletion interferes with myeloid progenitor progression and gene expression.16 However, ongoing studies have not directly addressed the specific regulatory role of Rheb1 in hematopoietic stem cells.
In this study, we observed that Rheb1 is an essential reg- ulator of hematopoietic development. Rheb1-deficient mice showed increased phenotypic HSCs, immature neu- trophils in bone marrow (BM), and splenomegaly. These phenotypes are reminiscent of the hematopoiesis seen in MPNs. Rheb1-deficient HSCs were defective in their abil- ity to reconstitute the blood tissue and differentiate into normal neutrophils. Interestingly, low Rheb expression was associated with poor survival in acute myeloid leukemia (AML) patients. Thus, our data indicate that Rheb is critical for HSC function and may be involved in the initiation of myeloid proliferation-related diseases or MPN-like disorders.
Methods
Mice and genotyping
Rheb1fl/fl mice were kindly provided by Dr. Bo Xiao.17 Transgenic mice expressing Cre recombinase under the control of the Vav1 promoter (Vav1 Cre) were purchased from the Jackson Lab. The Rheb1fl/fl mice were crossed with Vav1-Cre mice to generate specif- ic deletion of Rheb1 in the hematopoietic system. All animal pro-
tocols were approved by the Institutional Animal Care and Use Committee (IACUC), the Institute of Hematology, and Blood Diseases Hospital (CAMS/PUMC). All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize mouse suffering.
Flow cytometry analysis
Peripheral blood (PB) was obtained from either the tail veins or retro-orbital bleeding of mice. Red blood cells (RBCs) were lysed by ammonium chloride-potassium bicarbonate buffer before staining. BM cells were flushed out from tibias, femurs, and ilia by a 25-gauge needle with PBS supplemented with 2% fetal bovine serum (FBS) and 20 mM EDTA (abbreviated as PBE). Cells were stained with antibodies purchased from either eBioscience or BD Bioscience. To analyze intracellular proteins, 3x106 BM cells were labeled with surface antibodies, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X100, then washed 2 times with 1 mL cold PBE. Finally, the cells were resuspended with cold PBS supplemented with 25% FBS, and intracellularly stained with anti- bodies: p-S6 (Ser24/244), p-4EBP1 (Thr37/46). Cells were analyzed by BD Canto II flow cytometer. FlowJo software was used to ana- lyze the results.
LKS transplant and analysis
Whole BM cells (WBMCs) were obtained and Lin– cells were sorted using mouse lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s instruction. LKSs were stained as mentioned above and sorted by BD Influx flow cytometer; 200 LKSs (CD45.1) together with 5x105 whole BM cells (WBMCs) (CD45.2) were injected intravenously into lethally irradiated recip- ient mice (CD45.2). The reconstitution of PB cells was analyzed every four weeks post transplantation. The recipient mice were sacrificed at four months after transplantation. The self-renewal and differentiation capacities of donor-derived HSCs derived from BM were then analyzed.
Competitive bone marrow transplantation and analysis
Whole BM cells were isolated from the tibias, femurs and ilia of 8-week old Rheb1fl/fl (CD45.1) or Rheb1Δ/Δ mice (CD45.1). 5x105 Rheb1Δ/Δ WBMCs (CD45.1) together with 5x105 WBMCs (CD45.2) were intravenously injected into the lethally irradiated recipient mice (CD45.2). Then, the reconstituted PB cells were analyzed every four weeks after transplantation.
Lineage– cell homing assay
Whole BM cells were obtained, and LKS+ cells (CD45.1) were sorted by flow cytometry. LKS+ cells were cultured with CFSE at 37°C for 8 minutes (min). The reaction was then terminated with 10% FBS at 4°C for 2 min and washed two times with cold PBS. LKS+ cells (2x106) were intravenously injected into lethally irradiat- ed (9.5 Gy) recipient mice (CD45.2). The recipient mice were sac- rificed at 17 hours (h) or 24 h after transplantation. CFSE+ cells in BM of recipient mice were analyzed by FACS.
Histological and pathological analysis
To examine the histology of the BM neutrophils, the neu- trophils were sorted with CD11b and Ly-6G from BM, then cytospun and stained with Wright-Giemsa solution. For patholog- ical analysis, BM, spleen, liver or lung were fixed with 4% forma- lin, embedded in paraffin, sectioned, and stained with hema- toxylin and eosin.
In vitro bacterial killing assay
Escherichia coli (strain 19138; ATCC, Manassas, VA, USA) were
cultured overnight, suspended in PBS at an OD600 of 0.10, and
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haematologica | 2019; 104(2)