Dickkopf-1 promotes hematopoietic regeneration via direct and niche-mediated mechanisms

Journal name:
Nature Medicine
Volume:
23,
Pages:
91–99
Year published:
DOI:
doi:10.1038/nm.4251
Received
Accepted
Published online

Abstract

The role of osteolineage cells in regulating hematopoietic stem cell (HSC) regeneration following myelosuppression is not well understood. Here we show that deletion of the pro-apoptotic genes Bak and Bax in osterix (Osx, also known as Sp7 transcription factor 7)-expressing cells in mice promotes HSC regeneration and hematopoietic radioprotection following total body irradiation. These mice showed increased bone marrow (BM) levels of the protein dickkopf-1 (Dkk1), which was produced in Osx-expressing BM cells. Treatment of irradiated HSCs with Dkk1 in vitro increased the recovery of both long-term repopulating HSCs and progenitor cells, and systemic administration of Dkk1 to irradiated mice increased hematopoietic recovery and improved survival. Conversely, inducible deletion of one allele of Dkk1 in Osx-expressing cells in adult mice inhibited the recovery of BM stem and progenitor cells and of complete blood counts following irradiation. Dkk1 promoted hematopoietic regeneration via both direct effects on HSCs, in which treatment with Dkk1 decreased the levels of mitochondrial reactive oxygen species and suppressed senescence, and indirect effects on BM endothelial cells, in which treatment with Dkk1 induced epidermal growth factor (EGF) secretion. Accordingly, blockade of the EGF receptor partially abrogated Dkk1-mediated hematopoietic recovery. These data identify Dkk1 as a regulator of hematopoietic regeneration and demonstrate paracrine cross-talk between BM osteolineage cells and endothelial cells in regulating hematopoietic reconstitution following injury.

At a glance

Figures

  1. Deletion of Bak and Bax in Osx+ BM cells radioprotects hematopoietic stem and progenitor cells.
    Figure 1: Deletion of Bak and Bax in Osx+ BM cells radioprotects hematopoietic stem and progenitor cells.

    (a) Mean percentages of Osx+ cells, as measured by flow cytometry, within Osx-labeled BM cells (Osx–cherry+) and Osx-unlabeled cells (Osx–cherry) from 5-d-old (n = 4 mice/group) and 8-week-old (n = 8 mice/group) Osx–Cherry reporter mice. *P < 0.001, **P < 0.001. (b) Left, representative FACS plots showing the percentage of Osx+ cells in CD45 BM cells in Osx–Cre;Bak1−/−;BaxFL/+ (BAX FL/+) and Osx–Cre;Bak1−/−;BaxFL/− (BAX FL/−) mice at day +3 following 500 cGy TBI. Right, the mean percentage of Osx+CD45 BM cells (n = 4 mice/group). *P = 0.004. (c) Representative images of H&E-stained femurs from BAX FL/+ (left) and BAX FL/− (middle) mice at day +7 following 500 cGy TBI (40×; scale bars, 100 μm) and scatter plot of BM cell counts for mice in each group (n = 11 mice/group) (right). Horizontal lines represent means. *P = 0.009. (d) Mean numbers of BM KSL cells (*P = 0.03) (left) and SLAM+KSL cells (*P = 0.04) (right) in BAX FL/+ and BAX FL/− mice at day +7 following 500 cGy TBI (n = 11 mice/group). (e) Mean numbers of BM CFCs at day +7 (n = 21 assays/group). WBM, whole bone marrow; CFU-GM, colony-forming unit–granulocyte monocyte; BFU-E, burst-forming unit–erythroid; CFU-GEMM, colony-forming unit–mix. *P < 0.0001. (f) Mean PB WBC (*P = 0.01) (left), neutrophil (*P = 0.02) (middle) and lymphocyte (*P = 0.02) (right) counts at day +7 (FL/+, n = 14 mice; FL/−, n = 13 mice). (g) Donor (CD45.2+) cell engraftment over time in recipient CD45.1+ mice that were transplanted with 1 × 106 BM cells from BAX FL/+ or BAX FL/− mice and 1 × 105 competing, non-irradiated host BM cells. *P = 0.03, *P = 0.03, *P = 0.02 and *P = 0.04 for 4, 8, 12 and 20 weeks, respectively. Myeloid (Mac1/Gr1; *P = 0.04), B cell (B220; *P = 0.04) and T cell (CD3; *P = 0.03) engraftment levels at 20 weeks are shown (4, 8 and 12 weeks: FL/+, n = 14 mice; FL/−, n = 15 mice; 20 weeks: FL/+, n = 9 mice; FL/−, n = 13 mice). (h) Mean levels of donor CD45.2+ cell and lineage engraftment in secondary recipient CD45.1+ mice at 20 weeks following competitive transplantation with BM cells from primary mice (FL/+, n = 7 mice; FL/−, n = 9 mice). CD45.2+, *P = 0.04; B220+, *P = 0.03; CD3+, *P = 0.004. (i) Mean Dkk1 levels in the BM of non-irradiated or irradiated (500 cGy) BAX FL/+ and BAX FL/- mice. *P < 0.001 and **P < 0.001 (NI FL/+, n = 5 mice; NI FL/-, n = 9 mice; IRR FL/+, n = 8 mice; IRR FL/-, n = 7 mice). (j) Mean expression of Dkk1 in BM osteolineage cells in BAX FL/− mice relative to that in BAX FL/+ mice (n = 6 mice/group). *P = 0.02. (k) Dkk1 expression in VE-cadherin+ BM ECs and Osx+ BM cells in mice before (0 cGy; VE-cadherin+, n = 3 mice; Osx+, n = 4 mice) and 48 h after 500 cGy TBI (n = 6 mice/group). Dkk1 expression levels in all groups were normalized to that in VE-cadherin+ BM ECs at 48 h. *P = 0.03 and **P = 0.003. Throughout, P values were derived by using a two-tailed Student's t-test.

  2. Dkk1 promotes hematopoietic regeneration in vitro and in vivo.
    Figure 2: Dkk1 promotes hematopoietic regeneration in vitro and in vivo.

    (a) Mean numbers of CFCs recovered from 7-d cultures of irradiated (300 cGy) BM KSL cells that were treated with TPO, SCF and Flt-3 ligand (TSF)-containing medium (medium), with or without 500 ng/ml Dkk1 (n = 3 assays/group). *P = 0.003. (b) Left, mean percentage of donor CD45.2+ cell engraftment levels in recipient CD45.1+ mice over time following competitive transplantation with the progeny of 1 × 104 irradiated (300 cGy) BM KSL cells that were cultured with or without 500 ng/ml Dkk1 for 7 d. *P = 0.01, *P = 0.03, *P = 0.03, *P = 0.01 for 4, 8, 12 and 16 weeks, respectively. Right, mean percentages of donor myeloid (*P = 0.02), B cell (*P = 0.01) and T cell (*P = 0.01) engraftment at 16 weeks in recipient mice (n = 17 mice/group). (c) Dkk1 concentrations in the BM of mice at baseline, following 500 cGy treatment, and at 1 h and 24 h following administration of 10 μg Dkk1 administration (BL6 non-irradiated (NI), n = 5 mice; BL6, n = 5 mice; Dkk1 (1 h), n = 6 mice; Dkk1 (24 h), n = 5 mice). *P < 0.001, **P < 0.001. (d) Scatter plots of PB WBCs (left), neutrophils (middle) and platelet (right) counts at day +21 from mice irradiated with 800 cGy and then treated with saline or Dkk1 (saline, n = 5 mice; Dkk1, n = 12 mice). *P = 0.008, *P = 0.02, *P = 0.01; by Mann–Whitney U test. (e) Left, representative images of H&E-stained femurs from saline-treated (left) and Dkk1-treated (middle) mice at day +21 following treatment with 800 cGy (40×; scale bars, 100 μm). Right, mean BM cell counts (n = 5 mice/group). *P = 0.02. (f) Left, representative FACS plots showing the percentage of BM KSL cells in saline-treated and Dkk1-treated mice at day +21 following treatment with 800 cGy TBI. Middle and right, the mean percentage of KSL cells (*P = 0.02) (middle) and KSL cell numbers (*P = 0.008) (right) (n = 5 mice/group). P values determined by Mann–Whitney U test. (g) Mean numbers of BM CFCs at day +21 following treatment with 800 cGy TBI (n = 6 assays/group). *P = 0.04. (h) Survival of saline-treated (4/15) or Dkk1-treated (13/14) mice following treatment with 800 cGy TBI (27% versus 93%; P = 0.0004 by log-rank test). (i) Mean numbers of CFCs in C57BL/6 mice at 12 weeks after 600 cGy TBI and treatment with 10 μg Dkk1 or saline, subcutaneously, every other day through day +21 (n = 5 assays/group). *P = 0. 001. Throughout, unless otherwise noted, P values were derived by using a two-tailed Student's t-test.

  3. Inhibition or deficiency of Dkk1 in Osx-expressing BM cells suppresses hematopoietic regeneration.
    Figure 3: Inhibition or deficiency of Dkk1 in Osx-expressing BM cells suppresses hematopoietic regeneration.

    (a) PB WBCs, neutrophils and lymphocytes in mice treated with anti-Dkk1 or control IgG at day +10 following 500 cGy TBI. *P = 0.0004, *P < 0.0001, *P = 0.002, respectively (n = 10 mice/group). (b) Left, representative images of H&E-stained femurs stained with IgG (top) or anti-Dkk1 (bottom) at day +10 (40×; scale bars, 50 μm). Right, BM cell counts at day +10 (n = 5 mice/group). *P < 0.001. (c) Mean KSL cell numbers at day +10 (n = 5 mice/group). *P = 0.007. (d) Survival analysis of mice irradiated with 750 cGy TBI and treated with anti-Dkk1 or IgG (n = 12 mice/group). P = 0.006. (e) The hematopoietic profiles (PB WBCs (*P < 0.001), neutrophils (*P = 0.001) and lymphocytes (*P = 0.007)) of Osx–Cre;Dkk1FL/+ (Dkk1FL/+) and Osx–Cre;Dkk1+/+ (Dkk1+/+) mice at day 10 after irradiation with 500 cGy TBI and of irradiated Dkk1FL/+ mice that were treated every other day with Dkk1 through day +10 (Dkk1FL/+ + Dkk1) and evaluated at day +10 (Dkk1+/+, n = 9 mice; Dkk1FL/+, n = 10 mice; Dkk1FL/+ + Dkk1, n = 10 mice). (f) BM cell counts at day +10 (Dkk1+/+, n = 9 mice; Dkk1FL/+, n = 10 mice; Dkk1FL/+ + Dkk1, n = 10 mice). *P < 0.001. (g) BM KSL cell numbers at day +10 (Dkk1+/+, n = 7 mice; Dkk1FL/+, n = 10 mice; Dkk1FL/+ + Dkk1, n = 10 mice). *P = 0.002, **P = 0.03. (h) Mean numbers of BM CFCs at day +10 (Dkk1+/+, n = 6 assays; Dkk1FL/+, n = 6 assays; Dkk1FL/+ + Dkk1, n = 3 assays). *P = 0.008, **P = 0.005. (i) Mean concentrations of PB WBCs (*P = 0.001), neutrophils (*P < 0.001) and lymphocytes (*P < 0.001) in Osx–Cre;Dkk1+/+ mice (Dkk1+/+) and 8-week-Osx–Cre;Dkk1FL/+ mice (8-week-Dkk1FL/+) at day +10 following 500 cGy TBI (Dkk1+/+, n = 11 mice; 8-week-Dkk1FL/+, n = 7 mice). (j) Left, representative images of H&E-stained femurs from Dkk1+/+ (top) and 8-week-Dkk1FL/+ (bottom) mice at day +10 (40×; scale bars, 50 μm). Right, mean BM cell counts per femur (Dkk1+/+, n = 11 mice; 8-week-Dkk1FL/+, n = 7 mice). *P < 0.001. (k) Mean BM KSL cells at day +10 (Dkk1+/+, n = 7 mice; 8-week-Dkk1FL/+, n = 6 mice). *P = 0.01. (l) Mean BM CFCs at day +10 (Dkk1+/+, n = 6 assays; 8-week-Dkk1FL/+, n = 3 assays). *P = 0.005. Throughout, P values were derived by using a two-tailed Student's t-test.

  4. Dkk1 suppresses HSC senescence following irradiation and induces EGF secretion by BM ECs.
    Figure 4: Dkk1 suppresses HSC senescence following irradiation and induces EGF secretion by BM ECs.

    (a) The percentage of MitoSOX+ cells in non-irradiated CD34 KSL cells (NI) and at 24 h after 300 cGy TBI without treatment (medium) or with treatment with 500 ng/ml Dkk1, 100 ng/ml Wnt3a or 200 ng/ml Wnt5a; treatment was initiated 15 min before irradiation (n = 4 cell cultures/group). Medium included TPO, SCF and Flt-3 ligand. *P < 0.001, **P < 0.001. (b) The percentage of phospho-p38-positve cells in CD34 KSL cells at 24 h after 300 cGy irradiation (NI, n = 8 cultures; medium, n = 7 cultures; Dkk1, n = 8 cultures; Wnt3a, n = 8 cultures; Wnt5a, n = 7 cultures). *P < 0.001, **P < 0.001. (c) The percentage of SA–β-gal+ CD34 KSL cells at 24 h after 300 cGy irradiation (n = 4 cultures/group). *P < 0.001, **P < 0.001. (d) Left, p16 gene expression in BM CD34 KSL cells at 24 h after 300 cGy irradiation in the indicated treatment groups. *P = 0.005, **P = 0.02 (n = 4 cultures/group). Right, p16 expression in BM KSL cells at 24 and 48 h after 500 cGy TBI in mice treated without (saline) or with 10 μg Dkk1 (n = 4 cultures/group). 24 h, *P < 0.001, **P < 0.001; 48 h, *P = 0.02, **P = 0.02. (e) Mean percentages of SA–β-gal+ BM KSL cells in non-irradiated mice and in mice at 12 weeks following 600 cGy TBI and treated with saline or 10 μg Dkk1 every other day through day +21 (NI, n = 4 mice; saline, n = 7 mice; Dkk1, n = 7 mice). *P = 0.04, **P = 0.02. (f) Left, representative histograms of Axin2–LacZ+ cells within BM CD34 KSL cells, KSL cells and lin cells in mice at 24 h after 500 cGy TBI in the treatment groups shown. Right, the mean percentage of Axin2+ cells in each cell population at 24 h following 500 cGy TBI with and without Dkk1 treatment (n = 4 mice/group). CD34 KSL cells, *P = 0.009, **P = 0.01; KSL cells, *P < 0.001, **P = 0.004; lin cells, *P < 0.001, **P = 0.002. (g) Left, mean levels of PTN in cultures of BM ECs (n = 5 cultures/group). Middle, mean levels of EGF in BM ECs in response to 500 ng/nl Dkk1 treatment (*P < 0.001; BMEC, n = 5 cultures; BMEC + Dkk1, n = 10 cultures). Right, mean levels of EGF in C57BL/6 mice over time following 500 cGy TBI and treatment with saline or 10 μg Dkk1 every other day through day +14 (n = 5 mice/group) (day +10, *P = 0.03; day +14, *P = 0.02). (h) Mean values for PB WBCs, neutrophils, BM cell counts and BM KSL cells at days +10 and +14 following 500 cGy TBI and treatment with saline or 10 μg Dkk1, with and without 10 μg/g erlotinib (n = 5 mice/group). WBCs: day +10, *P < 0.001;day +14, *P = 0.002; neutrophils: day +10, *P < 0.001; day +14, *P = 0.01; BM cell counts: day +14, *P = 0.02; BM KSL cells: day +14, *P = 0.01. Throughout, P values were derived by using a two-tailed Student's t-test.

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Author information

  1. These authors contributed equally to this work.

    • Heather A Himburg &
    • Phuong L Doan

Affiliations

  1. Division of Hematology and Oncology, Department of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California, USA.

    • Heather A Himburg,
    • Mamle Quarmyne,
    • Xiao Yan,
    • Joshua Sasine,
    • Liman Zhao,
    • Jenny Kan,
    • Katherine A Pohl,
    • Evelyn Tran &
    • John P Chute
  2. Division of Hematologic Malignancies and Cellular Therapy, Duke University, Durham, North Carolina, USA.

    • Phuong L Doan,
    • Nelson J Chao &
    • Jeffrey R Harris
  3. Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, USA.

    • Mamle Quarmyne &
    • Xiao Yan
  4. Department of Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California, USA.

    • Grace V Hancock
  5. Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California, USA.

    • John P Chute
  6. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California, USA.

    • John P Chute

Contributions

J.P.C. conceived of and designed the study; H.A.H. designed and performed the majority of the experiments and analyzed data; P.L.D. and J.R.H. performed experiments and analyzed data; M.Q., X.Y., J.S., L.Z., G.V.H., J.K., K.A.P. and E.T. performed experiments; N.J.C. contributed to the design and interpretation of the study; and H.A.H. and J.P.C. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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