Rosiglitazone Promotes Bone Marrow Adipogenesis to Impair Myelopoiesis under Stress

Abstract

Objective: The therapeutic use of thiazolidinediones (TZDs) causes unwanted hematological side effects, although the underlying mechanisms of these effects are poorly understood. This study tests the hypothesis that rosiglitazone impairs the maintenance and differentiation of hematopoietic stem/progenitor cells, which ultimately leads to hematological abnormalities.

Methods: Mice were fed a rosiglitazone-supplemented diet or a normal diet for 6 weeks. To induce hematopoietic stress, all mice were injected once with 250 mg/kg 5-fluorouracil (5-Fu) intraperitoneally. Next, hematopoietic recovery, hematopoietic stem/progenitor cells (HSPCs) subsets, and myeloid differentiation after 5-Fu treatment were evaluated. The adipogenesis induced by rosiglitazone was assessed by histopathology and oil red O staining. The effect of adipocytes on HSPCs was studied with an in vitro co-culture system.

Results: Rosiglitazone significantly enhanced bone marrow adipogenesis and delayed hematopoietic recovery after 5-Fu treatment. Moreover, rosiglitazone inhibited proliferation of a granulocyte/monocyte progenitor (GMP) cell population and granulocyte/macrophage colony-stimulating factor (GM-CSF) colonies, although the proliferation and mobilization of Lin-c-kit+Sca-1+ cells (LSK) was maintained following hematopoietic stress. These effects could be partially reversed by the selective PPARγ antagonist BADGE. Finally, we demonstrated in a co-culture system that differentiated adipocytes actively suppressed the myeloid differentiation of HSPCs.

Conclusion: Taken together, our results demonstrate that rosiglitazone inhibits myeloid differentiation of HSPCs after stress partially by inducing bone marrow adipogenesis. Targeting the bone marrow microenvironment might be one mechanism by which rosiglitazone impairs stress-induced hematopoiesis.

Fig 1. Normal homeostatic hematopoiesis in rosiglitazone-treated mice.

(A) Adipocytes in tibia BM sections from rosiglitazone-treated and control mice (HE staining, scale bar 200 μm).(B) Adipocyte counts per mm² in tibia BM sections from both groups of mice. (C) Expression of PPARγ2 and adiponectin in BMMNCs from the two groups of mice. (D-F) No significant differences in peripheral blood(PB) counts were observed in rosiglitazone-treated mice compared to the control mice. (G) A decreased bone marrow mononuclear cell(BMMNC) count was observed in rosiglitazone-treated mice compared to control mice, but this difference was not statistically significant(P = 0.066). The data are presented as the means ± SD from three independent experiments.

Fig 2. Delayed hematopoietic recovery and increased mortality after rosiglitazone treatment.

(A)Adipocytes in tibia BM sections from the ROSI, the ROSI+BADGE and the control groups following 5-Fu treatment (HE staining, scale bar 200 μm). (B)Adipocyte counts per mm² in tibia BM sections from the three groups of mice. (C)Expression of PPARγ2 and adiponectin in BMMNCs from the three groups of mice after 5-Fu treatment. (D) Six-week treatment with rosiglitazone significantly delayed the recovery of white blood cells after 5-Fu treatment, and this effect was reversed by administering the PPARγ antagonist BADGE. (E-F)No significant differences in platelet and Hb levels were observed in rosiglitazone-treated mice compared to control mice. (G) The survival of mice was assessed daily. The data represent the means±SD, n = 3 in A–C, n = 4 in D-F, n = 8 in G. *P<0.05 compared to CTL,***P<0.001 compared to CTL, ^##P<0.01 compared to ROSI).

Fig 3. The in vivo effect of rosiglitazone treatment on LSK cells in bone marrow, peripheral blood and spleen under stress conditions.

(A) Schematic of the experiment. In brief, the mice were fed a diet with or without rosiglitazone for 4 weeks. Then, they received a single dose of 250 mg/kg 5-Fu and continued their rosiglitazone-enriched or normal diet for two weeks. Two weeks after chemotherapy, the bone marrow, spleen and PB cells were isolated and used for flow cytometric analysis or a CFU assay. (B) Flow cytometric analyses of the LSK and HPC populations in the BM 14 days after 5-Fu. (C)The number of BMMNCs in rosiglitazone-treated mice was dramatically reduced compared to control mice 14 days after 5-Fu treatment. (D) The absolute number of HPC and LSK cells in the BM 14 days after 5-Fu treatment. (E)Cell cycle analysis of LSK in BM. (F)Flow cytometric analysis of the proportion of LSK cells in the PB and spleen 14 days after 5-Fu treatment. The data are presented as the means±SD of three independent experiments, *P<0.05 compared to CTL, ***P<0.001 compared to CTL, ^##P<0.01 compared to ROSI.

Fig 4. Rosiglitazone impairs differentiation of myeloid progenitors in vivo.

(A-C) Flow cytometric analyses of HPC subsets in the BM 14 days after 5-Fu.(D) The colony-forming potential of BMMNCs 14 days after 5-Fu treatment. (E) RNA was extracted from BMMNCs from the CTL, ROSI and ROSI+BADGE group, and samples were analyzed for the expression of C/EBPα and PU.1. The data are presented as the means±SD of three independent experiments, *P<0.05 compared to CTL,**P<0.01 compared to CTL,^#P<0.05compared to ROSI,^##P<0.01 compared to ROSI.

Fig 5. The effect of rosiglitazone-treated stromal cells on myeloid differentiation in HSPCs.

(A) C3H10T1/2 cells and M2-10B4 were treated with 10 μM rosiglitazone in the presence or absence of BADGE(20μM). After 12 days of culture, the cells were fixed, and adipogenic differentiation was determined by Oil red O staining of the lipid droplets(scale bar 100 μm). (B, C) C3H10T1/2 cells and M2-10B4 were treated with 10 μM rosiglitazone in the presence or absence of BADGE(20μM) for 12 days. Then, the cells were washed twice with PBS followed by co-culture with Lin^- cells. After 3 days, colony-forming cell (CFC) assays (BFU-E and CFU-GM) were performed to determine the colony-forming viability of co-cultured Lin- cells. (D) The proportion of Gr-1⁺/CD11b⁺ cells was significantly decreased after co-culturing Lin^- cells with the rosiglitazone-treated C3H10T1/2 or M2-10B4 cell line. Furthermore, these effects were partially reversed by treating the stromal cells with BADGE. (The data are presented as the means±SD of three independent experiments, *P<0.05 vs. control, **P<0.01 vs. control, ^#P<0.05vs. ROSI group).

Fig 6. Rosiglitazone has no effect on hematopoietic progenitor cells in vitro.

Lin^- cells from the BM of 5-Fu-treated mice were selected and cultured in the presence or absence of rosiglitazone for 7 days, and the cell proliferation, cell phenotype and colony-forming potential of Lin^- cells were assayed. The data are presented as the means±SD of three independent experiments, **P<0.01 compared to control, ***P<0.001compared to control.