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The TRAP/Mediator complex, the metazoan counterpart of the yeast Mediator complex, is master transcriptional regulatory complex composed of approximately 30 subunits. It was originally isolated as a thyroid hormone receptor (TR)-associated protein (TRAP) complex that mediates TR-activated transcription from DNA templates in vitro and probably acts in vivo after the action of other receptor-interacting coactivators involved in chromatin remodeling. The TRAP220/MED1 subunit of the TRAP/Mediator complex is proposed to act on a variety of major and specific biological events, including growth, differentiation and homeostasis, through physical interaction with nuclear receptors. The vitamin D receptor (VDR) and retinoic acid receptor (RAR), coupled with retinoid X receptor (RXR), are nuclear receptors which have important roles for monopoiesis and granulopoiesis, respectively. In this study, we present the functional role of TRAP220/MED1 in nuclear receptor-mediated monopoiesis and granulopoiesis. Since TRAP220 knockout (Trap220-/-) mice were mortalities during the early embryonic period before definitive hematopoiesis within the hepatic primordia becomes dominant, the function of TRAP220/MED1 in adult hematopoiesis was mostly unknown. However, these embryos appeared to have normal composition of nucleated erythroid cells. Therefore, the E9.0 yolk sac-derived hematopoietic precursor cells were used to differentiate into definitive hematopoietic colony forming units within the methylcellulose blood cell culture. The number of monocytic colonies (CFU-M) was significantly lower in knockouts than in wild type controls, while the numbers of other types of colonies (CFU-GEMM, CFU-GM, CFU-G and CFU-E) were comparable. Hence, TRAP220/MED1 appeared to be indispensable for optimal monocytic differentiation. Next, the HL-60 acute promyelocytic leukemia cells were used to elucidate directly and mechanistically the roles of TRAP220/MED1 in RAR- and VDR-dependent differentiation of the hematopoietic precursor cells into granulocytic and monocytic lineage cells. The expression of the TRAP220/MED1 subunit as well as other TRAP/Mediator subunits was induced when the cells were treated with their ligands, all-trans retinoic acid and 1,25-dihydroxyvitamin D3. Flow cytometric analyses showed that HL-60 cells, wherein TRAP220/MED1 was down-regulated, did not differentiate efficiently into monocytes and granulocytes by stimulation with 1,25-dihydroxyvitamin D3 and all-trans retinoic acid, correspondingly. The expression of direct target genes of VDR or RAR, as well as the differentiation marker genes, was low in the knockdown cells by stimulation with these ligands. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA)- and dimethylsulphoxide (DMSO)-mediated monocytic and myeloid differentiation, which bypasses nuclear receptor-mediated signaling pathways, was not affected in knockdown cells. Collectively, these results indicated an indispensable role of TRAP220/MED1 in the optimal VDR- and RAR-mediated myelomonocytic differentiation processes in mammalian hematopoiesis.

Casein kinase I (CKI) ε is involved in cytokine-induced hematopoietic cell differentiation by directly interacting with the suppressor of cytokine signaling 3 (SOCS3) and/or β-catenin. Here, we show that CKIε plays an important role in hematopoietic cell survival through modifying Phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Introduction of wild-type (WT)-CKIε into murine interleukin-3 (IL-3)-dependent myeloid progenitor 32D cells increased the sensitivity to genotoxic stress, such as γ-irradiation, anti-cancer drug and cytokine-deprivation, whereas kinase-negative (KN)-CKIε suppressed it. While IL-3-induced Akt phosphorylation at Ser473 was sustained by KN-CKIε, it was attenuated by WT-CKIε. Similarly, in human promyelocytic leukemia HL-60 cells, the increase of phosphorylated Akt induced by all-trans retinoic acid was accompanied by the decrease of CKIε expression. A specific inhibitor for CKIε, CKI-7 also increased the phospho-Akt as well as phospho-PTEN (Ser380/Thr382/383; an inactive form of PTEN). Functional expression of CKIε activated PTEN by their physical association, following the decrease of phospho-Akt. The present studies suggest that the expression of CKIε downregulates PI3K/Akt signaling through PTEN, increasing the sensitivity to apoptotic signals. Taken together with the fact that CKIε is ubiquitously expressed in normal hematopoietic cells, CKIε might be critical for prevention against genotoxic stress-induced leukomogenesis. In other words, CKIε could be a candidate of the tumor suppressor in hematological malignancies as well as cancer.