The Transcriptional Mediator Subunit MED1/TRAP220 in Stromal Cells Is Involved in Hematopoietic Stem/Progenitor Cell Support through Osteopontin Expression

Abstract

MED1/TRAP220, a subunit of the transcriptional Mediator/TRAP complex, is crucial for various biological events through its interaction with distinct activators, such as nuclear receptors and GATA family activators. In hematopoiesis, MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow ( The specialized microenvironmental niches in the bone marrow (BM), namely, the osteoblastic (or endosteal) and vascular niches, host and interface with hematopoietic stem cells (HSCs) and are the sites where their size and fate are strictly regulated (15, 29; reviewed in references 1, 16, 28, 30, and 34). HSCs and their niches produce diverse molecules, whose interactions control HSC self-renewal and differentiation. In the osteoblastic niche, almost 75% of the HSCs are in a quiescent (slowly cycling or G 0 ) state. In a physiological condition, HSCs migrate from the osteoblastic niche toward the vascular niche, enter the cell cycle, and undergo symmetric cell division or asymmetric division, accompanied by differentiation and final maturation. In this manner, a defined set of mature differentiated progeny is continuously produced without HSC depletion.The transcriptional Mediator complex, originally isolated as a thyroid hormone receptor-associated protein (TRAP) complex and subsequently identified as a mammalian counterpart of the yeast Mediator complex (i.e., a subcomplex of the RNA polymerase II holoenzyme), appears to serve as a bridge between diverse activators and the general transcriptional machinery (reviewed in references 4, 13, 18, and 21). This complex contains approximately 25 polypeptides, among which the MED1/TRAP220 subunit is responsible for specific binding of the complex to several activators, which include nuclear receptors (13), GATA family members (22, 27), C/EBP␤ (20), and BRCA1 (33). Mediator conveys the specific signals of the activators to the recruited general transcriptional machinery to activate transcription by direct communication between MED1 and the activators (5).Through the interaction with MED1, nuclear receptors are involved in various hematocytic differentiations. For example, the vitamin D receptor (VDR) and retinoic acid receptor (RAR) are members of the nuclear hormone receptor superfamily, whose interaction with MED1 is crucial for liganddependent monopoiesis and granulopoiesis, respectively, as well as for peroxisome proliferator-activated receptor ␥ (PPAR␥)-mediated adipogenesis (7, 31). GATA-1, for which MED1 was recently shown to be a specific coactivator, mediates erythropoiesis through its interaction with MED1 (27). However, as Med1 null mice die early during embryogenesis (12,22), it is difficult to determine the physiological role of MED1 in BM hematopoiesis in vivo.Osteopontin (OPN), an acidic glyc...

Periostin supports hematopoietic progenitor cells and niche-dependent myeloblastoma cells in vitro

“…Stable lines of Med1 +/+ p53 −/− and Med1 −/− p53 −/− MEFs in C57BL6 background are described elsewhere [8]. MEFs, mouse BM-derived MSCs obtained from GIBCO, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum (FBS).…”

“…MEFs, mouse BM-derived MSCs obtained from GIBCO, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum (FBS). The OP-9 cells, distributed by RIKEN BRC through the National Bio-Resource of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and MS-5 cells [15] were maintained as described [8]. The niche-dependent MB-1 myeloblastoma cells were maintained by coculturing with mitomycin C (MMC)-treated OP-9 cells as described [16,17].…”

“…We previously reported that Med1 −/− mouse embryonic fibroblasts (MEFs) have an attenuated ability to support hematopoietic progenitor cells (HPCs) relative wild-type MEFs, and that the attenuated expression of full-length osteopontin or FGF7 in Med1 −/− MEFs is responsible for the observed phenotype. Thus, osteopontin and FGF7, produced by BM mesenchymal stromal cells, have been identified as important niche factors [8,9]. …”

Mediator Subunits MED1 and MED24 Cooperatively Contribute to Pubertal Mammary Gland Development and Growth of Breast Carcinoma Cells

“…The cell viability was assessed by trypan blue exclusion. For DNA synthesis, the incorporation of BrdU into cells in 24-well plates, after purging for 6 h, was measured by using a cell proliferation enzyme-linked immunosorbent assay (ELISA) with BrdU (chemiluminescence) (Roche) (38).…”

“…The cDNAs for activators (20 ng of human ER␣ [hER␣], human PR␤ [hPR␤], and Gal4-fused vitamin D receptor [VDR] and VP16), cloned into the cytomegalovirus (CMV) promoter-driven mammalian expression vector pcDNA3.1 (Invitrogen), together with the firefly luciferase reporter pGL3 (Promega), containing either 3ϫ ER-responsive element (ERE), 4ϫ PR-responsive element (PRE), or 5ϫ Gal4-binding sites (100 ng), were transfected with the Renilla control luciferase vector (5 ng) into MEFs using Lipofectamine (Invitrogen). After 48 h, the reporter activities were measured using the dual-luciferase reporter assay system (Promega) and normalized to the control Renilla luciferase activity (38).…”