Critical role of Jak2 in the maintenance and function of adult hematopoietic stem cells

Abstract

Jak2, a member of the Janus kinase family of nonreceptor protein tyrosine kinases, is activated in response to a variety of cytokines, and functions in survival and proliferation of cells. An activating JAK2V617F mutation has been found in most patients with myeloproliferative neoplasms, and patients treated with Jak2 inhibitors show significant hematopoietic toxicities. However, the role of Jak2 in adult hematopoietic stem cells (HSCs) has not been clearly elucidated. Using a conditional Jak2 knockout allele, we have found that Jak2 deletion results in rapid loss of HSCs/progenitors leading to bone marrow failure and early lethality in adult mice. Jak2 deficiency causes marked impairment in HSC function, and the mutant HSCs are severely defective in reconstituting hematopoiesis in recipient animals. Jak2 deficiency also causes significant apoptosis and loss of quiescence in HSC-enriched LSK (Lin(-)Sca-1(+)c-Kit(+)) cells. Jak2-deficient LSK cells exhibit elevated reactive oxygen species levels and enhanced p38 MAPK activation. Mutant LSK cells also show defective Stat5, Erk, and Akt activation in response to thrombopoietin and stem cell factor. Gene expression analysis reveals significant downregulation of genes related to HSC quiescence and self-renewal in Jak2-deficient LSK cells. These data suggest that Jak2 plays a critical role in the maintenance and function of adult HSCs.

Keywords: Apoptosis; Conditional knockout; Hematopoietic stem cells; JAK2 kinase; Proliferation; Self-renewal; Signal transduction.

Figure 1. Deletion of Jak2 leads to fatal BM failure and loss of HSCs/progenitors

(A) The expression of Jak2 in the BM of Jak2fl/fl (control) and MxCre;Jak2fl/fl (Jak2-deficient) mice was determined by immunoblotting at 4 weeks after pI;pC injection. Erk2 was used as a loading control. (B) Kaplan-Meier survival analysis of MxCre;Jak2fl/fl (n=24) and littermate control (Jak2fl/fl) (n=24) mice after pI;pC treatment. MxCre;Jak2fl/fl mice have a median survival of 36 days after 1^st pI;pC injection; *P< 0.05, log-rank test. (C) H&E staining of the BM sections (200×) demonstrates pancytopenia and severe aplasia in Jak2-deficient BM. (D) Flow cytometric analysis of LSK compartment (Lin⁻Sca1⁺c-kit⁺) and myeloid progenitors including CMP (Lin⁻Sca1⁺c-kit⁺CD34⁺FcγRII/III^lo), CLP (Lin⁻CD127⁺Sca1⁺c-kit⁺), GMP (Lin⁻Sca1⁺c-kit⁺CD34⁺FcγRII/III^hi), and MEP (Lin⁻Sca1⁺c-kit⁺CD34⁻FcγRII/III⁻) in the BM from control or Jak2-deficient (MxCre;Jak2fl/fl) mice (n=10) at 4 weeks after pI:pC injection. The percentages and absolute numbers of LSK, CLP, CMP, GMP, and MEP are shown in bar graphs as mean ± SEM. *P< 0.05. (E) Flow cytometric analysis of LT-HSC (Lin⁻Sca1⁺c-kit⁺CD34⁻CD135⁻), ST-HSC (Lin⁻Sca1⁺c-kit⁺CD34⁺CD135⁻), and MPP (Lin⁻Sca1⁺c-kit⁺CD34⁺CD135⁺) in the BM from control or Jak2-deficient (MxCre;Jak2fl/fl) mice at 4 weeks after pI:pC injection (n=7). Representative contour plots are shown in the top. The percentages and absolute numbers of LT-HSC, ST-HSC, and MPP are shown in bar graphs as mean ± SEM. *P< 0.05. (F) Flow cytometric analysis of SLAM-HSC (Lin⁻Sca1⁺c-kit⁺CD150⁺CD41⁻CD48⁻) in the BM from control or Jak2-deficient (MxCre;Jak2fl/fl) mice at 4 weeks after pI:pC injection (n=5). Representative contour plots are shown on the top. The percentages and absolute numbers of SLAM-HSC are shown in bar graphs as mean ± SEM; *P< 0.05. (G, H) Hematopoietic progenitor colony assays. Total BM cells (2×10⁴) (G) or sorted LSK cells (100) (H) from control or Jak2-deficient (MxCre;Jak2fl/fl) mice were plated in methylcellulose medium MethoCult 3434 (StemCell Technologies) with cytokines. BFU-E, CFU-GM and CFU-GEMM colonies were scored 7–8 days after plating. To assess CFU-E colonies, BM cells were plated in methycellulose medium in the presence of Epo. CFU-E colonies were scored 3 days after plating. To assess CFU-Mk colonies, BM cells were plated in collagen-based MegaCult media with IL-6, IL-11, and Tpo. Colonies were counted after 7–8 days. Note that deletion of Jak2 significantly reduces hematopoietic progenitor colonies in the total BM or sorted LSK cells from MxCre;Jak2fl/fl mice (n=5). *P< 0.05. (I) Cell autonomous BMT assay. BM cells (1×10⁶ cells) from uninduced MxCre;Jak2fl/fl or littermate control (Jak2fl/fl) mice (CD45.2⁺) were transplanted into lethally irradiated CD45.1 recipients. Deletion of Jak2 was induced into the donor-derived hematopoietic cells of recipient animals by injecting pI;pC four weeks after BMT. Note that donor-derived (CD45.2⁺) HSC (LSKCD150⁺CD41⁻CD48⁻) was significantly reduced in recipients of Jak2-deficient BM (n=5, *P< 0.05). (J) BM cells (1×10⁶ cells) from CD45.1 WT mice were transplanted into lethally-irradiated CD45.2 control (Jak2fl/fl) or MxCre;Jak2fl/fl recipients. Four weeks after BMT to allow establishment of steady-state hematopoiesis, Jak2 was deleted from non-hematopoietic cells in the mutant recipients with 5 doses of pI;pC injection. Note that CD45.1⁺ WT donor-derived HSCs (LSKCD150⁺CD41⁻CD48⁻) were not reduced upon induction of Jak2 deletion in mutant recipients (n=3).

Figure 2. Defective stem cell function in Jak2-deficient mice

(A) Non-competitive BMT assay. Different doses of BM cells from control (1×10⁵–1×10⁶ cells) or MxCre;Jak2fl/fl (1×10⁵–2×10⁶ cells) mice were transplanted into lethally irradiated CD45.1 recipient mice in absence of competitor cells. Note that Jak2-deficient BM cells failed to reconstitute hematopoiesis in recipient animals even when transplanted 20-times more mutant BM cells than control BM cells. (B) Competitive reconstitution assay with Jak2 deletion before BMT. Total BM cells from pI;pC induced control or MxCre;Jak2fl/fl (CD45.2) mice were transplanted into lethally irradiated CD45.1 recipient mice in competition with BM from CD45.1 WT mice, at different ratios (1:1 or 10:1) of donor versus competitor. The percentages of donor derived (CD45.2⁺) myeloid cells (Gr-1⁺), B cells (B220⁺), and T cells (TCRβ⁺) in peripheral blood at different time points after transplantation are shown as mean ± SEM. (n=5, *P< 0.05). (C) The percentages of donor-derived (CD45.2⁺) LSK in the BM of the recipient animals are shown at 12 weeks after transplantation (n=5). (D) Competitive reconstitution assay with Jak2 deletion after BMT. Total BM cells from uninduced MxCre;Jak2fl/fl or littermate control (CD45.2) mice were transplanted into lethally irradiated CD45.1 recipient mice in competition with BM from CD45.1 WT mice at 1:1 ratio. Four weeks after BMT, recipient mice were treated with 5 doses of pI;pC to induce Jak2 deletion after hematopoietic reconstitution. The percentages of donor derived (CD45.2⁺) myeloid cells (Gr-1⁺), B cells (B220⁺), and T cells (TCRβ⁺) in peripheral blood was measured every four weeks after pI;pC injection. (E) The percentages of donor-derived (CD45.2⁺) LSK in the BM of the recipient animals at 20 weeks after transplantation (16 weeks after pI;pC injection) are shown as mean ± SEM. (n=5, *P< 0.05). (F) Survival curves of secondary transplanted animals. CD45.2⁺ (donor-derived) cells from primary transplanted mice (as in Fig. 2D) were FACS sorted 12 weeks after pI;pC injection, and injected into lethally irradiated CD45.1 secondary recipient mice (5×10⁵ CD45.2⁺ cells/recipient) (n=5). Note that Jak2-deficient cells failed to engraft secondary recipients and all secondary recipients that had received Jak2-deficient cells died within 2 weeks after transplantation.

Figure 3. Reduced quiescence and increased apoptosis in Jak2-deficient LSK cells

(A) Control or Jak2-deficient mice were injected with BrdU and sacrificed 4 hours later. Representative FACS plots (in the top panel) illustrate BrdU/DAPI staining in LSK cells. Bar graphs in the bottom panel show percentages of BrdU⁺ LSK cells in G₀/G₁, S and G₂/M. Data from five independent experiments are shown as mean ± SEM. *P< 0.05. (B) Flow cytometric analysis of Hoechst and Pyronin Y staining on LSK cells from control and Jak2-deficient mice BM. Representative contour plots are shown on the top panel. Bar graphs in the bottom panel show percentages of LSK cells in G₀, G₁ and S/G₂/M. Data from five independent experiments are shown as mean ± SEM. *P< 0.05. (C) Apoptosis in LSK cells from control or Jak2-deficient mice was determined using Annexin V and DAPI staining. Representative contour plots are shown on the top. Bar graphs (in the bottom) represent percentages of apoptotic LSK cells in control or Jak2-deficient mice. Data from five independent experiments are shown as mean ± SEM. *P< 0.05.

Figure 4. Elevated ROS levels and increased p38 MAPK activation in Jak2-deficient LSK cells

(A) LSK cells from control (Jak2fl/fl) and Jak2-deficient mice were stained with CM-H₂DCFDA to measure intracellular ROS. Jak2-deficient LSK cells showed increased levels of ROS compared with control LSK. Representative FACS plots are shown on the left. Mean values ± SEM are shown on the right (n=3, *P< 0.05). (B) Flow cytometric analysis for phospho-p38 MAPK in LSK cells. MACS-enriched Lin- cells from control or Jak2-deficient mice BM were starved for 1 hour in serum-free media at 37°C. Cells were fixed, permeabilized, and stained with appropriately conjugated antibodies against LSK surface markers and Alexa488 conjugated phospho-p38 MAPK. Representative FACS plots on intracellular phospho-p38 MAPK staining in LSK cells are shown on the left. The fold change in mean fluorescent intensity (MFI) of phospho-p38 MAPK in Jak2-deficient LSK is shown in bar graphs (on the right) as mean ± SEM (n=4, *P< 0.05). (C) NAC treatment of Jak2-deficient mice BM partially rescued the defects in hematopoietic reconstitution. Lin⁻ BM cells from control or Jak2-deficient mice (CD45.2⁺) were treated with PBS or NAC ex vivo for 48 hours in StemSpan medium (Stem Cell Technologies) containing SCF (50 ng/ml) and Tpo (50 ng/ml) and then 1×10⁶ cells were transplanted in the presence of 2×10⁵ helper BM cells (CD45.1⁺) into lethally irradiated CD45.1 recipient mice. The percentages of donor-derived (CD45.2⁺) LSK in the BM of recipient animals were determined at 4 weeks after transplantation by flow cytometry. Representative contour plots are shown on the top panel. Bar graphs (in the bottom) show percentages of donor-derived (CD45.2⁺) LSK cells in recipient mice that had received PBS-treated or NAC-treated Jak2-deficient BM or control BM (n=4 in each group, *P< 0.05).

Figure 5. Signaling defects in Jak2-deficient LSK cells

(A) Lin⁻ BM cells from control or Jak2-deficient mice were starved for 1 hour in serum-free media at 37°C before they were either left untreated or stimulated with 50ng/ml Tpo for the indicated time. Cells were fixed, permeabilized, and stained with appropriately conjugated antibodies against LSK surface markers and Alexa488 conjugated phospho-stat5, phospho-Erk1/2 or phospho-Akt antibodies. Representative histograms are shown on the top panels, and the fold change in mean fluorescent intensity (MFI) in phospho-stat5, phospho-Erk1/2 or phospho-Akt in Jak2-deficient LSK cells are shown in bar graphs (in the bottom panels) as means ± SEM (n=3, *P< 0.05). (B) Representative histograms on Mpl (TPO receptor) expression in control or Jak2-deficient LSK cells are shown (top panel), and the fold change in mean fluorescent intensity (MFI) in Mpl expression from four independent experiments are shown (in the bottom panel) as means ± SEM. No significant (ns) difference was observed in Mpl expression between control and Jak2-deficient LSK cells. (C) Lin⁻ BM cells from control or Jak2-deficient mice were starved for 1 hour in serum-free media at 37°C before they were either left untreated or stimulated with 50ng/ml SCF for the indicated time. Cells were fixed, permeabilized, and stained for LSK surface markers and phospho-stat5, phospho-Erk1/2 or phospho-Akt as in panel A. Representative histograms are shown on the top panels, and the fold change in mean fluorescent intensity (MFI) in phospho-stat5, phospho-Erk1/2 or phospho-Akt in Jak2-deficient LSK cells are shown in bar graphs (in the bottom panels) as means ± SEM (n=3, *P< 0.05). (D) Relative expression of Kit in SLAM-HSC (Lin⁻Sca1⁺CD150⁺CD41⁻CD48⁻). Representative histograms are shown on the top panels, and the fold change in mean fluorescent intensity (MFI) in Kit expression is shown (in the bottom panel) as means ± SEM (n=4 in each group, *P< 0.05). Note that Kit expression was significantly reduced in Jak2-deficient HSCs.

Figure 6. Gene expression profile of Jak2-deficient LSK cells

(A) Gene-set enrichment analyses (GSEAs) were performed on microarray data obtained from control and Jak2-deficient LSK cells. Enrichment plots of selected gene sets from GSEA analysis with normalized enrichment score (NES) and false discovery rate (FDR) are shown. (B) Ingenuity pathway analysis (IPA) of microarray data revealed that most significantly affected molecular and cellular functions associated with Jak2 deficiency were cell death/survival, cell morphology, cellular development, cell cycle, cell growth, cellular function and maintenance, reflecting abnormal cell cycle and survival in Jak2-deficient HSCs. (C) Heat map showing differential expression of a selected list of genes related to HSC maintenance and function in control and Jak2-deficient LSK cells. (D) Relative expression of Jak2, c-Jun, Pim1, Gfi-1, Mecom, Pbx1, Foxo3, Atm, p21 mRNA was determined by quantitative real-time PCR and normalized with 18S expression. Asterisks indicate significant differences by Student t test with P< 0.05. (E) Ectopic expression of c-Jun only partially rescues the defects in hematopoietic progenitor colony formation in Jak2-deficient LSK cells. Lin⁻ BM cells from control or Jak2-deficient mice were transduced with lentiviruses expressing GFP alone or c-Jun-IRES-GFP or Jak2-IRES-GFP in StemSpan H3000 plus BIT9500 medium (StemCell Technologies) containing 50ng/ml SCF and 50ng/ml TPO. GFP-positive LSK cells were sorted using FACS and plated (1 × 10⁴ cells/dish) in methylcellulose medium containing complete cytokines (M3434; StemCell Technologies). Progenitor colonies were counted 7–8 days after plating. Results are expressed as percentage of controls (n=3; *P< 0.05). Note that ectopic expression of wild-type Jak2 almost completely rescued whereas c-Jun expression only partially rescued the defects in hematopoietic progenitor colony formation in Jak2-deficient LSK cells.