The multi-protein complex TRAP/Mediator is a subcomplex of RNA polymerase II holoenzyme and acts as the end-point integrator of a variety of activators and intracellular signalings. The Mediator activates transcription in concert with a group of recruited general transcription initiation factors. Among the circa 25 subunits, the MED1/TRAP220 subunit was originally demonstrated by us as a key component that acts as a ligand-dependent nuclear receptor-specific coactivator, and proved to play an important role in retinoic acid-dependent granulocytic and 1,25-dihydroxyvitamin D3-dependent monocytic differentiation of both normal hematopoietic precursor and acute promyelocytic leukemia cells, via retinoic acid receptor (RAR) and vitamin D receptor (VDR), respectively. MED1 was recently re-identified as a specific coactivator for GATA family activators and found to function in the process of GATA-1-mediated normal erythroblast differentiation. In this study, we analyzed the role of MED1 in erythroid differentiation of malignant erythroblastic (leukemic) cells. We used K562 human erythroleukemia cells as a model since GATA-1-mediated erythroid differentiation of K562 is efficiently induced with hemin. When K562 cells were exposed to 50nM hemin for 5 days, 44% of cells became positive for benzidine staining and the expression of the erythroid lineage-specific (and GATA-1-targeted) genes such as β-globin, γ-globin, porphobilinogen deaminase (PBGD) and 5-aminolevulinate synthase (ALAS-E) were strongly induced. The Mediator subunits including MED1 were also induced strongly together with these erythroid-specific genes in this process. Since the precedent study has demonstrated that Mediator subunits are induced during nuclear receptor-mediated myelomonopoiesis, and since genes whose products have physiological significance are often induced, Mediator might be employed in GATA-1-mediated erythroid differentiation of erythroleukemia cells as well. Then we focused on the GATA-1-specific coactivator MED1 among the Mediator subunits. In order to analyze the role of MED1, we first reduced the expression of endogenous MED1 in K562 cells with the MED1 knockdown small hairpin RNA (shRNA) expression vector. When the transiently knocked down cells (approximately 25% of the control) were treated with hemin, the number of benzidine-positive cells were half of the control in 3 days, indicating that hemin-induced erythroid differentiation was strongly inhibited. Further, the expression of direct target genes for GATA-1 such as β-globin, γ-globin, PBGD and ALAS-E were significantly reduced by approximately 70% in knockdown cells. Moreover, when the knockdown cells were cultured for 3 days without hemin, the basal expression levels of the erythroid lineage-specific genes such as β-globin, γ-globin and PBGD were significantly reduced by approximately 30%. We next asked if overexpressed MED1 might enhance erythroid differentiation. Since the MED1 domain near the N-terminus (a.a. 622 to 701) interacts with GATA-1 and that the N-terminal domain that contains two LxxLL nuclear receptor recognition motifs (NR box; aa. 592 to 703) is sufficient for in vitro and most in vivo transcription events, we tested the N-terminal truncation of MED1, N-MED1 (aa. 1 to 703), for overexpression. When N-MED1 was stably expressed in K562 cells after selection with G418, MED1 expression was boosted at least 2.2 folds of the control transfectant cells. When these cells were treated with hemin, benzidine-positive differentiated cells were 3-fold reduced and the expression of the erythroid differentiation marker genes (above) were approximately 2-fold increased in N-MED1 cells after one day. However, after 3 days, the number of benzidine-positive cells and the expression of erythroid marker genes in N-MED1 cells were saturated and caught up by the control cells. Thus, it appears that overexpressed MED1 enhances the erythroid differentiation in K562 cells. Together with the finding that GATA-1 is physically associated with MED1 and that MED1 is co-occupied with GATA-1 in the enhancer region, these results indicate that MED1 functions as a GATA-1-specific coactivator in erythroid differentiation of not only normal erythroblasts but erythroleukemic cells. MED1 also appears to be a key coactivator for two distinct lineages of normal and malignant hematopoiesis, namely myelomonopoiesis and erythropoiesis.
1984 The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures: No relevant conflicts of interest to declare.
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