Pivotal role for glycogen synthase kinase-3 in hematopoietic stem cell homeostasis in mice

Abstract

Hematopoietic stem cell (HSC) homeostasis depends on the balance between self renewal and lineage commitment, but what regulates this decision is not well understood. Using loss-of-function approaches in mice, we found that glycogen synthase kinase-3 (Gsk3) plays a pivotal role in controlling the decision between self renewal and differentiation of HSCs. Disruption of Gsk3 in BM transiently expanded phenotypic HSCs in a betta-catenin-dependent manner, consistent with a role for Wnt signaling in HSC homeostasis. However, in assays of long-term HSC function, disruption of Gsk3 progressively depleted HSCs through activation of mammalian target of rapamycin (mTOR). This long-term HSC depletion was prevented by mTOR inhibition and exacerbated by betta-catenin knockout. Thus, GSK-3 regulated both Wnt and mTOR signaling in mouse HSCs, with these pathways promoting HSC self renewal and lineage commitment, respectively, such that inhibition of Gsk3 in the presence of rapamycin expanded the HSC pool in vivo. These findings identify unexpected functions for GSK-3 in mouse HSC homeostasis, suggest a therapeutic approach to expand HSCs in vivo using currently available medications that target GSK-3 and mTOR, and provide a compelling explanation for the clinically prevalent hematopoietic effects observed in individuals prescribed the GSK-3 inhibitor lithium.

Figure 1. Gsk3 depletion expands HSCs and HPCs in primary transplants.

(A) Irradiated mice were reconstituted with BM after transduction with lentivirus with or without Gsk3-rnai. Peripheral blood was examined 20 weeks after transplantation; numbers within histograms indicate percent GFP⁺ cells. Shown is 1 representative of 5 similar experiments; similar results were obtained with Gsk3-rnai-C4. (B) GFP⁺ myeloid cells (Gr1⁺CD11b⁺) in peripheral blood for 10 control and 9 Gsk3-rnai-C2 recipients after BM transplantation (BMT; arrow). (C) Immunoblots for GSK-3α/β and β-catenin in BM from primary recipients 16 weeks after transplantation. Data represent independent replicates from 6 control and 6 Gsk3-rnai recipients. (D) Percent GFP⁺ LSK cells in control and Gsk3-rnai-C2 primary recipients. (E) Absolute number of GFP⁺ LSK, LSK CD34^–Flk2^–, and LSK CD34⁺Flk2^– cells. (F) Representative FCM showing GFP⁺ cells in the HSC-containing LSK fraction (red gate) for control and Gsk3-rnai-C2 primary recipients. (G) Representative FCM using SLAM markers; the difference between control and Gsk3-rnai was significant (P < 0.05). Numbers in F and G indicate percent cells within gates. (H) Colony formation using GFP⁺ cells plated in methylcellulose with cytokines and scored for CFU-C (see Supplemental Figure 1). Data represent mean colonies per well performed in duplicate groups for 5 mice per construct repeated in 3 separate experiments. *P < 0.05 versus respective control value.

Figure 2. Gsk3 knockdown increases cycling of the HSC-enriched LSK cell population.

(A) To assess cell cycle status of the HSC-enriched LSK population, sorted GFP⁺ LSK and GFP⁺ LSK Flk2^– cells from primary recipients of control and Gsk3-rnai 4 months after BM transplantation were stained with Hoechst and Pyronin and analyzed by FCM. Representative FACS data are shown for control versus Gsk3-rnai-C2. (B) At 4 months after BM transplantation, primary recipients of control and Gsk3-rnai were fed BrdU in the drinking water for 7 days. Sorted GFP⁺ LSK Flk2⁺ and Flk2^– cells were stained with BrdU-APC antibody and PI to analyze BrdU incorporation. Representative FACS data are shown for control versus Gsk3-rnai-C2. Similar results were obtained by BrdU versus 7-AAD staining (BD). Percent cells are shown for the indicated gates and quadrants. In A and B, lower left gate represents G₀, upper left represents G₁, and upper right represents S, G₂, and M phases of the cell cycle, as shown in the diagram in A.

Figure 3. Gsk3-depleted HSCs are functionally deficient.

(A) Limiting dilution experiments were performed with 4 doses (x axis) of GFP⁺ test BM from control and Gsk3-rnai primary recipients (4 donors per group) combined with a fixed 2 × 10⁵ unlabeled competing cells transplanted into groups of at least 5 recipients per dose. Chimerism at 4 months after transplantation for each dose is represented as the percentage of GFP⁺ cells in BM for control and Gsk3-rnai. (B) Percent donor-derived immunophenotypic HSCs/HPCs (as GFP⁺ LSK cells) in the 1 × 10⁶ test cell group 4 months after transplantation. (C) Absolute number of donor-derived immunophenotypic HSCs/HPCs (as GFP⁺ LSK cells) in the 1 × 10⁶ test cell group 4 months after transplantation.

Figure 4. Gsk3 knockdown depletes HSCs in serial BM transplants.

(A) Noncompetitive serial transplants were performed by transplanting 2 × 10⁵ sorted GFP⁺ cells from primary recipients of control or Gsk3-rnai transduced BM into lethally irradiated recipients (10 mice per group). Survival of secondary recipients receiving control or Gsk3-rnai BM is shown as a Kaplan-Meier plot. (B) Percent HSC-containing LSK fraction in control and Gsk3-rnai secondary recipients. (C) Absolute number of GFP⁺ LSK, LSK CD34^–Flk2^–, and LSK CD34⁺Flk2^– cells in control and Gsk3-rnai secondary recipients. (D) Representative FCM data, presented as the distribution of CD34^–Flk2^–, CD34⁺Flk2^–, and CD34⁺Flk2^–, which immunophenotypically correspond to LT-HSCs, ST-HSCs, and MPPs in the LSK population, from control and Gsk3-rnai secondary recipients. Percent cells are shown for the indicated gates. (E) Colony formation assay with sorted GFP⁺ cells from control and Gsk3-rnai secondary recipient BM was performed and scored as in Figure 1 using GFP⁺ BM from 5 control and 5 Gsk3-rnai mice. (F) The frequencies of common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) cells were measured by detection of CD16/32 and CD34 expression in the lineage^–sca-1^–c-kit⁺ gated population. The common lymphoid progenitor (CLP) fraction was measured as CD127⁺ cells in the lineage^–sca-1^loc-kit^lo gate. (G) Lethally irradiated mice were reconstituted with 4 × 10⁵ sorted GFP⁺ BM cells from secondary recipients of vector or Gsk3-rnai transduced BM. The Kaplan-Meier survival curve shows the survival of tertiary recipients of BM from control or Gsk3-rnai mice. (H) Absolute number of immunophenotypic HSCs/HPCs, as LSK cells, in control and Gsk3-rnai tertiary recipients. *P < 0.05.

Figure 5. β-catenin is required for the increase in HSCs/HPCs induced by Gsk3-rnai.

(A) BM cells were harvested from Mx-Cre;β-catenin^fl/fl mice with or without injection of polyI:polyC for 14 days, transduced with control or Gsk3-rnai carrying lentivirus, and transplanted into lethally irradiated recipient mice. After 4 months, percentage and absolute number of HSC-containing LSK fraction were compared among the 4 groups. (B) BM cells were harvested at 4 months from primary recipients of WT and Mx-Cre;β-catenin^fl/fl mice transduced with vector control or Gsk3-rnai lentivirus (from primary recipient mice in A) and transplanted into lethally irradiated secondary hosts. After 4 months, percentage and absolute number of HSC-containing LSK fraction were compared among the 4 groups. (C) Summary of serial transplantation data in WT versus β-catenin CKO mice. Shown is fold change in GFP⁺ LSK cells in recipients of Gsk3-depleted BM normalized to vector control, for otherwise WT primary, secondary, and tertiary recipients as well as for primary and secondary β-catenin CKO recipients. Survival in tertiary recipients of Gsk3/β-catenin–deficient BM was too low for statistical significance. *P < 0.05.

Figure 6. Increased HSCs/HPCs in rapamycin-treated recipients of Gsk3-depleted BM.

(A) BM was harvested from Mx-Cre;β-catenin^fl/fl mice treated with or without polyI:polyC, transduced with control or Gsk3-rnai lentivirus, and transplanted into irradiated recipients. After 4 months, GFP⁺ cells were sorted (pooled from 5 mice per group), and phospho–ribosomal protein S6 (p-S6) was detected in cell lysates by immunoblot. (B) Flow cytometric detection of phospho–ribosomal protein S6 with BM cells in A. (C) Irradiated mice were reconstituted with BM transduced with control vector or Pten-rnai. After 4 months, phospho–GSK-3 and phospho–ribosomal protein S6 were assessed in GFP⁺ cells by immunoblot. (D) NIH3T3 cells were infected for 3 days with control or Pten-rnai lentivirus, and phospho–GSK-3α/β in control and Pten-depleted cells was detected by immunoblot. (E) Noncompetitive serial transplants. GFP⁺ cells (2 × 10⁶) from primary recipients of control or Gsk3-rnai were transplanted into 10 irradiated recipients per group. After 1.5–2 months, secondary recipients were injected with rapamycin or vehicle every other day for 8 weeks. Percent GFP⁺ LSK cells was compared among the 4 groups. (F) Absolute number of GFP⁺ LSK cells as in E. (G) Colony formation with sorted GFP⁺ cells from E. (H) Kaplan-Meier plot showing survival of tertiary recipients transplanted with BM from control- and rapamycin-treated secondary recipients in E–G. Shown is control vector BM from secondary recipients treated with vehicle or rapamycin and Gsk3-rnai–infected BM from secondary recipients treated with vehicle or rapamycin transplanted to lethally irradiated tertiary recipients. *P < 0.05.

Figure 7. Gsk3b KO depletes HSCs in serial BM transplants.

Noncompetitive serial transplants were performed by transplanting 4 × 10⁵ fetal liver cells (CD45.2) from E17.5 WT, Gsk3b^+/–, and Gsk3b^–/– embryos into lethally irradiated recipient mice (CD45.1). (A and B) Reconstitution of peripheral blood, including B cells (B220⁺), T cells (CD3⁺), and myeloid cells (Mac-1⁺GR-1⁺), in primary (A) and secondary (B) recipients of WT, Gsk3b^+/–, and Gsk3b^–/– fetal liver cells. Secondary transplants were performed after 16 weeks of engraftment by pooling BM from 3–4 reconstituted primary recipients to transplant 4 × 10⁵ whole BM cells into lethally irradiated CD45.1 secondary recipients (10 hosts per genotype). (C and D) Percentage of LSK cells in CD45.2⁺ BM was compared among recipients of WT, Gsk3b^+/–, and Gsk3b^–/– fetal liver cells in primary (C) and secondary (D) hosts. *P < 0.05.

Figure 8. GSK-3 functions in 2 major pathways to regulate HSC self renewal and lineage commitment.

Inhibition of GSK-3 activates Wnt and mTOR signaling. In the canonical Wnt pathway, GSK-3 and β-catenin bind to the Axin complex, along with APC. GSK-3 phosphorylates β-catenin, targeting it for rapid destruction. Wnt binding to the Fz/Lrp receptor complex causes inhibition of GSK-3, which in turn stabilizes β-catenin and activates Wnt target genes that promote progenitor proliferation and self renewal. In PI3K/PTEN-regulated pathways, growth factors (GFs) bind to surface receptors and activate PI3K, leading to activation of Akt, whereas PTEN inhibits activation of Akt. Once activated, Akt phosphorylates and inhibits GSK-3. GSK-3 phosphorylates Tsc2, inhibiting the mTOR pathway. Thus, inhibition of GSK-3 activates mTOR and promotes proliferation and exit from the LT-HSC pool. Inhibition of GSK-3 thus activates distinct downstream signaling pathways that have opposing functions in HSC renewal and differentiation.