Vitamin K2 Modulates Differentiation and Apoptosis of Both Myeloid and Erythroid Lineages

Abstract 3993

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis eventually leading to maturation arrest and leukemic transformation. It is well-known that VK2 induces differentiation and apoptosis in acute myeloid leukemia (AML) cell lines such as HL-60 and U937. Based on the studies of AML cell lines, several clinical trials of VK2 therapy for MDS patients have been conducted, and they showed improvement of cytopenia and reductions in blastic cells. Interestingly, hematological improvement was also observed in MDS patients with low percentage of blasts, and a differentiation/apoptosis-inducing effect on blasts alone could not explain this fact. Thus, the effects of VK2 on primary hematopoietic progenitors were examined from the perspective of differentiation and apoptosis. Mobilized CD34-positive cells from peripheral blood were used for the examination of myeloid lineage cells, and were cultured in IMDM containing 20% FCS, 20 ng/mL rhSCF, 20 ng/mL rhIL-3, with or without VK2. VK2 induced significant increase of CD11b-positive cells on day 4 (35.8% ± 12.3% with 10 μM VK2 vs. 10.7% ± 1.9% without VK2, P=0.0034) and day 6 (42.7% ± 6.3% with 10 μM VK2 vs. 24.1% ± 8.6% without VK2, P=0.0235). CD14-positive cells also increased significantly on day 4 (8.0% ± 0.3% with 10 μM VK2 vs. 4.1% ± 1.5% without VK2, P=0.008). Furthermore, after treatment with VK2, mRNA expression levels of both C/EBPα and PU.1 were elevated in a dose-dependent manner, and a significant increase was shown at 10 μM of VK2 on day 6. These results indicate that VK2 promotes the differentiation of myeloid progenitors through the upregulation of transcriptional factors C/EBPα and PU.1. The effect of VK2 on the apoptosis of myeloid progenitors was also examined. VK2 increased the number of apoptotic cells determined by Annexin V assay transiently on day 4 (58.9% ± 6.3% with 10 μM VK2 vs. 36.1% ± 2.8% without VK2, P<0.0001), but no significant increase was found on day 6. Next, human erythroid colony forming cells (ECFCs) purified from peripheral blood were used for the examination of erythroid lineage cells. ECFCs were cultured in IMDM containing 15% FCS, 15% human AB serum, 2 U/ml rhEPO, 20 ng/mL rhSCF, 10 ng/mL rhIL-3 (depleted on day 3), with or without VK2 (added on day 3). In ECFCs, VK2 did not affect the expressions of transferrin receptor (TfR) and glycophorin A (GPA) or the expression level of β-globin mRNA. However, the expression of GATA-1 mRNA increased significantly on day 7 with 10 μM of VK2. VK2 seems to have the potential to promote the differentiation of ECFCs through the upregulation of transcriptional factor GATA-1, although this differentiating effect on ECFCs was much smaller than that on myeloid progenitors. Furthermore, VK2 exhibited an anti-apoptotic effect on day 7 ECFCs under erythropoietin (EPO) -depletion. The percentage of apoptotic cells after 24 hours of EPO-depletion, which was determined by Annexin V-positivity, was significantly reduced with VK2 at low concentrations (0.5-2 μM) (76.9% ± 4.7% with 1 μM VK2 vs. 88.3% ± 1.7% without VK2, P=0.0019). VK2 lost its anti-apoptotic effect at concentrations greater than 5 μM. This anti-apoptotic effect was not shown in erythroleukemic cell line AS-E2. Finally, the expression of steroid and xenobiotic receptor (SXR), which was recently identified as a receptor of VK2, on primary hematopoietic cells was examined. SXR was expressed on myeloid progenitors, but not on erythroid progenitors. SXR agonist rifampicin also upregulated the expressions of CD11b and CD14 on myeloid progenitors. The differentiation-promoting effect of VK2 on myeloid progenitors seems to be mediated partly through SXR signaling. These results indicate that the effect of VK2 varies by cell type. The major effect on myeloid progenitors was promoting differentiation, whereas its anti-apoptotic effect seemed to be dominant in erythroid progenitors. Although the detailed mechanism of VK2's effect on differentiation or apoptosis of hematopoietic progenitors remains unknown, the effect of VK2 therapy in MDS patients could be partly explained by these mechanisms.