Growth differentiation factor 15 contributes to marrow adipocyte remodeling in response to the growth of leukemic cells

Abstract

Background: The adipocyte remodeling, including of the morphological change, might indicate special pathological function. Our previous study found that the morphological remodeling of larger marrow adipocytes into small marrow adipocytes correlates with a poor prognosis for acute myeloid leukemia (AML) patients. However, the mechanisms contributed to the marrow adipocyte remodeling are still poorly understood.

Methods: GDF15 expression was analyzed by RT-qPCR and western blotting assays in the leukemic cells. The enhancing and antibody neutralization tests in vitro were employed to evaluate the effect of GDF15 on the morphology of mature adipocytes. CCK8 test was used to detect the proliferation of leukemic cells after co-cultivation with small marrow adipocytes. Flow cytometry was used to analysis the proportion of cell cycle of leukemic cells. Immunofluorescence staining and linear analysis were applied to verify the GDF15 expression and the relationship between GDF15 and small marrow adipocytes in AML patients.

Results: In this study, we found that leukemic cell lines not only expressed significantly higher growth differentiation factor 15 (GDF15) than the other three cytokines associated with adipocyte differentiation in RNA level but also secreted GDF15 factor. Furthermore, the in vitro experiments demonstrated that GDF15 was involved in the conversion of small marrow adipocytes from larger marrow adipocytes. Correspondingly, the leukemic cells proliferated more rapidly through regulating the cell cycle when co-cultured with GDF15-induced small marrow adipocytes. The immunofluorescence staining on the bone marrow sections of AML patients further exhibited that GDF15 was partly produced by leukemic cells. The positive correlation between the concentration of GDF15 in the marrow aspirates and the number and the volume of small marrow adipocytes might suggest the contribution of GDF15 in AML patients (r = 0.72, r = 0.67).

Conclusions: GDF15 secreted by leukemic cells was involved in the morphological remodeling of marrow adipocytes, which can in turn promote leukemic cell growth, indicating that GDF15 may be a promising treatment target for AML patients.

Keywords: Acute myeloid leukemia; Adipocyte remodeling; GDF15; Marrow adipocyte.

Fig. 1

The soluble cytokines secreted by leukemic cells contribute to the adipocyte remodeling. a BMSC-derived adipocytes cultured with HG-DMEM supplied with 10% FBS (Ctr) or with the conditioned medium (CM) of healthy mononuclear cells (MNC CM), primary leukemic cells (LC CM), THP-1 cell line (THP-1 CM), K562 cell line (K562 CM), HL-60 cell line (HL-60 CM) and Kasumi cell line (Kasumi CM) for 5 days. Adipocytes were stained by ORO. Both images are at a magnification of 400×. b The average area of adipocytes cultured with the CM of different AML cells were compared with the controls by using Image-Pro-Plus 5.1. *P < 0.05, **P < 0.01. c The content of adipocyte lipid-droplets in indicated groups was detected by OD values after ORO staining. *P < 0.05, **P < 0.01. d RT-qPCR analysis of adipogenic genes (FABP4, C/EBPα and PPARγ) in indicated groups. *P < 0.05, **P < 0.01. Results shown are from three independent experiments. Values shown are the mean ± SEM

Fig. 2

AML cell lines highly express GDF15. a RT-qPCR analysis of different cytokines associated with the regulation of adipogenesis in AML cell lines (K562, THP-1 and HL-60). GAPDH was used as a housekeeping gene. *P < 0.05, ***P < 0.001. b and c, RT-qPCR (b) and Western blotting (c) analysis of GDF15 in different cell lines (Kasumi, HL-60, THP-1, K562 and HEL). The densitometry values of protein expression changes were indicated. β-actin was used as an internal control for RT-qPCR and Western blotting analysis. d ELISA detection of GDF15 expression in the supernatant of THP-1 cells with different cell densities

Fig. 3

Contribution of GDF15 secreted by leukemic cells to the adipocyte remodeling. a Adipocytes treated with rhGDF15 (+GDF15) or neutralizing anti-GDF15 antibody (CM + anti-GDF15) or co-cultured with GDF15 knock down THP-1 cells (Ctr + THP-1 GDF15-KD) were stained with Alexa Fluor 493/503-conjugated BODIPY. Adipocytes cultured alone (Ctr) or cultured with the conditioned medium of THP-1 cells (CM) or co-cultured with negative control (Ctr + THP-1 GDF15-NC) were used as the controls respectively. Scale bar represents 100 μm. b The area of each adipocyte in each group was analyzed by using Image-Pro-Plus 5.1. *P < 0.05, **P < 0.01. c Adipocyte number in each group was analyzed by using Image-Pro-Plus 5.1. *P < 0.05, **P < 0.01. d The adipogenic gene (FABP4, PPARγ, C/EBPα) expression was detected by RT-qPCR analysis in each group. *P < 0.05, **P < 0.01, ***P < 0.001. Results shown are from three independent experiments. Values shown are the mean ± SEM

Fig. 4

GDF15-induced small adipocytes promote the AML cells growth by increasing the lipolysis. a RT-qPCR analysis of lipolytic genes (HSL and ATGL) in adipocytes treated with rhGDF15 or cultured alone. *P < 0.05. b The content of free fat acid (FFA) in the supernatant of adipocytes from indicated groups was detected using the colorimetric method. **P < 0.01. c CCK8 detection of the proliferation of THP-1 cells and K562 cells cultivated with conditioned medium of adipocytes (Ad-CM) or small adipocytes (sAd-CM) from the indicated two groups. The statistical difference was compared between sAd-CM group and Ad-CM group of these two leukemic cells. *P < 0.05, **P < 0.01. Results shown are from three independent experiments. Values shown are the mean ± SEM. d Representative images showed the cell cycle of THP-1 cells and K562 cells treated with Ad-CM or sAd-CM. e Western blotting analysis of Cyclin D1, CDK2 and P21 protein levels in THP-1 and K562 cells treated with Ad-CM or sAd-CM. β-actin and GAPDH protein were used as internal controls for Western blotting analysis

Fig. 5

The relationship of GDF15 expression and small marrow adipocytes in AML patients. a RT-qPCR analysis of GDF15 mRNA expression in BM from AML patients (n = 15) and the controls (n = 12). The results shown are from three independent experiments. *P < 0.05. b Western blotting analysis of GDF15 protein levels in BM from AML patients and the controls. The densitometry values of protein expression changes were indicated. β-actin protein was used as an internal control for Western blotting analysis. c and d Representative confocal images showed the expression of GDF15 and leukemic cell markers CD34 (c) or CD117 (d) in BM sections of AML patients. DAPI was used to stain the nuclei. White triangles showed the leukemic cells with GDF15+. White arrows showed the non-leukemic cells with GDF15. Scale bar represents 40 μm. e and f Scatter plot showed the positive correlation of small adipocyte volume (e) or small adipocyte number (f) with the level of GDF15 in BM of AML (n = 20, R = 0.6679, P = 0.0013, or n = 20, R = 0.7205, P = 0.003, Spearman correlation test)