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Nupr1 regulates the quiescence threshold of HSC
Nuclear protein transcription regulator 1 (NUPR1) is a member of the high-mobility group of proteins, which was first discovered in the rat pancreas during the acute phase of pancreatitis and was initially called p8.10 The same gene was discovered in breast cancer and was named Com1.11 NUPR1 has various roles, being involved in apoptosis, stress response, and cancer progression, depending on dis- tinct cellular contexts. In certain cancers, such as breast cancer, NUPR1 inhibits tumor cell apoptosis and induces tumor establishment and progression.12-15 In stark contrast, in prostate cancer and pancreatic cancer, NUPR1 has an inhibitory effect on tumor growth.16,17 There is accumulat- ing evidence that NUPR1 is a stress-induced protein: inter- ference of NUPR1 can upregulate the sensitivity of astro- cytes to oxidative stress;18 loss of it can promote resistance of fibroblasts to adriamycin-induced apoptosis;19 NUPR1 mediates cannabinoid-induced apoptosis of tumor cells;20 and overexpression of NUPR1 can negatively regulate MSL1-dependent histone acetyltransferase activity in Hela cells, which induces chromatin remodeling and relaxation allowing access of the repair machinery to DNA.21 Nonetheless, the potential roles of Nupr1, which is prefer- entially expressed in HSC among the hematopoietic stem and progenitor cells, in hematopoiesis remain elusive.
NUPR1 interacts with p53 to regulate cell cycle and apoptosis responding to stress in breast epithelial cells.19,22 p53 plays several roles in homeostasis, proliferation, stress, apoptosis, and aging of hematopoietic cells.23-27 Deletion of p53 upregulates HSC self-renewal but impairs the repopu- lating ability of these cells and leads to tumors.28 Hyperactive expression of p53 in HSC decreases the size of the HSC pool, and reduces engraftment and deep quies- cence.29-31 These findings support the essential check-point role of p53 in regulating HSC fate. Nonetheless, it is unknown whether NUPR1 and p53 coordinately regulate the quiescence of HSC.
Here, we used a Nupr1 conditional knockout model to investigate the consequences of loss of function of Nupr1 in the context of HSC. Nupr1 deletion in HSC led to the cells exiting from quiescence under homeostasis. In a com- petitive repopulation setting, Nupr1-deleted HSC prolifer- ated robustly and showed dominant engraftment over their wild-type counterparts. Nupr1-deleted HSC also expanded abundantly and preserved their stemness in vitro. The rescued expression of p53 by Mdm2+/- offset the effects introduced by loss of Nupr1 in HSC. Our studies reveal a new role and signaling mechanism of Nupr1 in regulating the quiescence of HSC.
Methods
Mice
Animals were housed in the animal facility of the Guangzhou Institutes of Biomedicine and Health (GIBH). Nupr1fl/fl mice were provided by Beijing Biocytogen Co., Ltd. CD45.1, Vav-cre, Mx1- cre, and Mdm2+/- mice were purchased from the Jackson Laboratory. All the mouse lines were maintained on a pure C57BL/6 genetic background. All experiments were conducted in accordance with experimental protocols approved by the Animal Ethics Committee of GIBH.
Hematopoietic stem cell cycle analysis
We first labeled the HSC with (CD2, CD3, CD4, CD8, Ter119, B220, Gr1, CD48)-Alexa Fluor700, Sca1-Percp-cy5.5, c-
kit-APC-cy7, CD150-PE-cy7, CD34-FITC and CD135-PE. The cells were then fixed using 4% paraformaldehyde. After wash- ing, the fixed cells were permeabilized with 0.1% saponin in phosphate-buffered saline together with Ki-67-APC staining for 45 min. Finally, the cells were resuspended in DAPI solution for staining for 1 h. The data were analyzed using Flowjo soft- ware.
Bromodeoxyuridine incorporation assay
Nupr1-/- mice and WT littermates were injected with 1 mg bro- modeoxyuridine (BrdU) on day 0. They were then allowed to drink water containing BrdU (0.8 mg/mL) ad libitum. On days 3, 4, and 5 after the injection of BrdU, four mice of each group were sacrificed. The rate of BrdU incorporation was analyzed by flow cytometry according to the BD PharmingenTM APC BrdU Flow Kit instructions.
Hematopoietic stem cell culture
The HSC culture protocol has been described elsewhere.32 Briefly, 50 HSC were sorted into fibronectin (Sigma)-coated 96- well U-bottomed plates directly and were cultured in F12 medi- um (Life Technologies), 1% insulin-transferrin- selenium- ethanolamine (ITSX; Life Technologies), 10 mM HEPES (Life Technologies), 1% penicillin/streptomycin/glutamine (P/S/G; Life Technologies), 100 ng/mL mouse thrombopoietin, 10 ng/mL mouse stem cell factor and 0.1% polyvinyl alcohol (P8136, Sigma-Aldrich). Half the medium was changed every 2-3 days, by manually removing medium by pipetting and replacing it with fresh medium, as indicated.
Limiting dilution assays
For limiting dilution assays,33 cells cultured for 10 days were transplanted into lethally irradiated C57BL/6-CD45.1 recipient mice, together with 2×105 CD45.1 bone-marrow competitor cells. Recipients were analyzed every 4 weeks. Limiting dilution analysis was performed using ELDA software.34 based on 1% peripheral blood multilineage chimerism as the threshold for positive engraftment.
Bone marrow competitive repopulation assay
One day before bone marrow transplantation, adult C57BL/6 recipient mice (CD45.1, 8-10 weeks old) were irradi- ated with two doses of 4.5 Gy (RS 2000, Rad Source) at a 4- hour interval. Bone marrow nucleated cells (BMNC; 2.5×105) from Nupr1-/- mice (CD45.2) and their WT (CD45.1) counter- parts were mixed and injected into irradiated CD45.1 recipi- ents by retro-orbital injection. Control BMNC (CD45.2), Mdm2+/-Nupr1-/- BMNC (CD45.2) or Mdm2+/- BMNC (CD45.2) were also mixed with the same number of competitors (CD45.1) and transplanted into recipients. For Nupr1fl/flMx1-cre transplantation, cre expression was induced through intraperi- toneal injection of polyinosinic-polycytidylic acid (pI-pC, 250 mg/mouse) every other day 1 week before transplantation. The same number (2.5×105) of Nupr1fl/flMx1-cre and WT (CD45.1) BMNC were mixed and transplanted into the lethally irradiat- ed CD45.1 recipients. Mx1-cre+ mice were taken as the exper- iment control. Mx1-cre and WT (CD45.1) BMNC (2.5×105) were used for the transplant control. The transplanted mice were maintained on trimethoprim-sulfamethoxazole-treated water for 2 weeks. For secondary transplantation, BMNC (1×106) were obtained from primary competitive transplanted recipients and injected into irradiated CD45.1 recipients (2 doses of 4.5 Gy, 1 day before transplantation). Donor-derived cells and hematopoietic lineages in peripheral blood were assessed monthly by flow cytometry.
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