Erythropoietin inhibits osteoblast function in myelodysplastic syndromes via the canonical Wnt pathway

Abstract

The effects of erythropoietin on osteoblasts and bone formation are controversial. Since patients with myelodysplastic syndromes often display excessively high erythropoietin levels, we aimed to analyze the effect of erythropoietin on osteoblast function in myelodysplastic syndromes and define the role of Wnt signaling in this process. Expression of osteoblast-specific genes and subsequent osteoblast mineralization was increased in mesenchymal stromal cells from healthy young donors by in vitro erythropoietin treatment. However, erythropoietin failed to increase osteoblast mineralization in old healthy donors and in patients with myelodysplasia, whereas the basal differentiation potential of the latter was already significantly reduced compared to that of age-matched controls (P<0.01). This was accompanied by a significantly reduced expression of genes of the canonical Wnt pathway. Treatment of these cells with erythropoietin further inhibited the canonical Wnt pathway. Exposure of murine cells (C2C12) to erythropoietin also produced a dose-dependent inhibition of TCF/LEF promoter activity (maximum at 500 IU/mL, -2.8-fold; P<0.01). The decreased differentiation capacity of erythropoietin-pretreated mesenchymal stromal cells from patients with myelodysplasia could be restored by activating the Wnt pathway using lithium chloride or parathyroid hormone. Its hematopoiesis-supporting capacity was reduced, while reactivation of the canonical Wnt pathway in mesenchymal stromal cells could reverse this effect. Thus, these data demonstrate that erythropoietin modulates components of the osteo-hematopoietic niche in a context-dependent manner being anabolic in young, but catabolic in mature bone cells. Targeting the Wnt pathway in patients with myelodysplastic syndromes may be an appealing strategy to promote the functional capacity of the osteo-hematopoietic niche.

Figure 1.

Different effects of erythropoietin on differentiation of osteoblasts from young and old healthy donors and patients with myelodysplastic syndromes. Human mesenchymal stromal cells from young healthy donors (A–B), MDS patients and age-matched healthy donors (C) were differentiated towards osteoblasts in the presence of various concentrations of erythropoietin (Epo). The mineralization was visualized with Alizarin red S staining and quantified after elution with cetylpyridinium chloride (A, C). Gene expression analysis of alkaline phosphatase (ALP) and osteoprotegerin (OPG) using real-time polymerase chain reaction in MSC from young healthy donors after treatment with 50 IU/mL Epo for 10 days (B). N=3–5. *P<0.05, **P<0.01, ns – not significant vs. control (CO).

Figure 2.

Erythropoietin inhibits canonical Wnt signaling, which is already suppressed in mesenchymal stromal cells from patients with myelodysplastic syndromes. (A) Human mesenchymal stromal cells from old healthy donors and MDS patients were compared using Wnt profiler polymerase chain reaction (white bars). Afterwards, osteoblasts from MDS patients were treated with 50 IU/mL erythropoietin (Epo) for 24 h and were compared to untreated cells (black bars). All genes presented in the bar graph were significantly regulated (P<0.05). N=3. (B) Murine myoblast C2C12 cell line was transfected with the Signal TCF/LEF Reporter and treated with increasing Epo concentrations for 24 h. Luciferase activity was assayed 24 h after treatment. N=4. *P<0.05, **P<0.01 vs. control.

Figure 3.

Activators of the canonical Wnt pathway can restore the attenuated osteoblastic differentiation of erythropoietin-treated mesenchymal stromal cells from patients with myelodysplastic syndromes. Human mesenchymal stromal cells from young healthy donors (A) and MDS patients (B) were differentiated towards osteoblasts in the presence of 50 IU/ml erythropoietin (Epo) with intermittent 100 ng/mL parathyroid hormne (PTH) or 25 mM lithium chloride (LiCl). The mineralization was visualized with Alizarin red S staining and quantified after elution with cetylpyridinium chloride. N=3–5. *P<0.05, **P<0.01, ***P<0.001 vs. control (CO).

Figure 4.

The hematopoietic support by erythropoietin-treated mesenchymal stromal cells is inhibited but can be restored by Wnt pathway activation. Human mesenchymal stromal cells (MSC) were pretreated with erythropoietin (Epo) 50 IU/mL and/or lithium chloride (LiCl) 25 mM for 7 days and co-cultured with freshly isolated CD34⁺ HSPC. After 7 days, flow cytometric analysis was performed for the non-adherent cells. (A) Number of CD34⁺ cells and (B) number of CD38⁺ cells. Each dot represents the percentage of positive-stained cells; each graph represents one donor/patient. (C) After 4 weeks of co-culture, the number of cobblestone area-forming cells (CAF-C) was determined in each well. A colony-forming unit (CFU) assay was performed in methylcellulose medium for 2 additional weeks and the colonies were classified under a microscope for HSPC co-cultured with (D) young healthy MSC, (E) old healthy MSC and (F) MDS MSC. N=3–5. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001