Abstract
The retinoid X receptor (RXR) acts as an obligate heterodimeric partner for multiple nuclear hormone receptors (NRs), including the retinoic acid receptor (RAR), thyroid receptor (TR), vitamin D receptor (VDR), and peroxisome proliferator-activated receptor (PPAR). Targeted disruption of RXRα in the mouse yields an embryonic lethal phenotype due to impairment of cardiac development. We have utilized a conditional knockout approach to investigate the roles of NR signaling in haematopoiesis. Bone marrow cells were isolated from a mouse homozygous for a targeted mutation in which exon IV of RXRα is flanked by loxP sites (RXRαfl/fl). This mutation permits normal expression of RXRα, but expression of cre recombinase results in excision of exon IV, abrogating expression of functional RXRα (RXRαko/ko). We employed a retrovirus to deliver cre to conditionally targeted haematopoietic cells. Lineage-depleted RXRαfl/fl bone marrow (BM) cells were transduced with a retrovirus that expresses a GFP-cre fusion, or with a control retrovirus expressing only EGFP. Transduced cells were isolated to >97% purity by FACS. The effect of RXRα disruption on haematopoiesis was assessed by in vitro assays and by transplantation into strain-matched lethally irradiated recipient mice. Progenitor assays performed in methylcellulose medium supplemented with haematopoietic growth factors revealed that GFP-cre - transduced (RXRαko/ko) grafts contain slightly fewer BFU-E and CFU-GM per 10,000 cells (60% and 80% of EGFP - transduced RXRαfl/fl cells, respectively). Long-term culture initiating cells (LTCIC) were enumerated for RXRαko/ko and RXRαfl/fl grafts. RXRα excision resulted in a moderate (25%) reduction in LTCIC. RXRαko/ko HSCs grown in suspension culture (IMDM supplemented with 10% foetal bovine serum, IL3, IL6, and kit ligand) for two weeks show reduced proportions of Mac1 positive (5% vs 27%) and Gr-1 positive (5% vs 12%) cells and strikingly increased CD117 positive cells (84% vs 49%). In vivo function of RXRαko/ko HSCs was evaluated by transplantation into lethally irradiated mice. Recipients were analyzed at 2, 4, and 6 weeks post-transplantation. Two weeks after transplantation, RXRαko/ko and RXRαfl/fl HSCs showed similar patterns of engraftment, with GFP-positive erythroid and myeloid cells found mainly in the spleen. At 4 weeks, recipients of RXRαfl/fl grafts showed significant BM engraftment of myeloid and erythroid lineages, while RXRαko/ko recipients exhibited minimal BM engraftment. At 6 weeks post-transplant, engraftment of RXRαfl/fl cells was well-established in both BM and spleen. RXRαko/ko HSCs showed minimal myeloid engraftment, but both spleen and BM were populated predominantly by Ter119 positive erythroid cells, which exhibit markedly dyserythropoietic morphology. This difference was reflected in peripheral blood counts; recipients of RXRαko/ko grafts were profoundly anaemic, thrombocytopenic, and neutropenic, and pronounced RBC polychromasia and poikilocytosis. These data indicate that disruption of RXRα in adult HSCs results in a modest reduction in early and committed progenitors in vitro, but profoundly disrupts ability to reconstitute haematopoiesis in a lethally irradiated recipient. Myeloid and megakaryocyte lineages do not engraft in recipients of RXRαko/ko HSCs. RXRαko/ko erythropoiesis is dysplastic and yields markedly abnormal erythrocytes. Further investigation is required to elucidate the multiple roles of RXRα in haematopoiesis.
Peroxisome Proliferator-Activated Receptors (PPARs) as Potential Inducers of Antineoplastic Effects in CNS Tumors
