Identification of the molecular requirements for an RARα-mediated cell cycle arrest during granulocytic differentiation

Abstract

Retinoids are potent inducers of cell cycle arrest and differentiation of numerous cell types, notably granulocytes. However the mechanisms by which retinoids mediate cell cycle arrest during differentiation remain unclear. We have used myeloid differentiation to characterize the molecular pathways that couple cell cycle withdrawal to terminal differentiation. Using primary cells from mice deficient for either the cyclin-dependent kinase inhibitor (CDKi) p27 Kip1 , the Myc antagonist Mad1, or both Mad1 and p27 Kip1 , we observed that signals mediated through reti- IntroductionThe roles of retinoids during hematopoiesis, particularly granulopoiesis, have been recognized for many years. Most strikingly, retinoids are powerful inducers of differentiation. 1 This effect is central to the use of all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL), a leukemia characterized by a chromosomal translocation always involving retinoic acid receptor ␣ (RAR␣). 2,3 Pharmacologic doses of ATRA are able to relieve the repression caused by the fusion proteins via directly binding and activating them and promoting cell cycle arrest and differentiation of the leukemic blasts. 3 However, the mechanisms by which ATRA mediates cell cycle arrest as well as differentiation remain unclear. An understanding of the molecular basis of this effect will provide important insights into how cell cycle withdrawal is coupled to terminal differentiation and how retinoids interact with the cell cycle.The effects of retinoid ligands are mediated through direct binding to the ligand-binding domain of RARs or retinoid X receptors (RXRs), members of the nuclear hormone receptor superfamily. 4 RARs heterodimerize with RXRs, and this heterodimer binds DNA at retinoic acid response elements (RAREs) in the regulatory sequences of target genes. When unliganded, RAR/RXR heterodimers bind RARE and form repressive complexes through binding SMRT-and NCo-R-containing corepressor complexes. 5 Ligand binding triggers the release of the corepressor complexes and coactivator complexes are recruited to the RAR/ RXR dimer and activate gene transcription. [6][7][8] Three isotypes of RARs and RXRs have each been identified: ␣, ␤, and ␥, with each RAR isotype expressed as a number of isoforms. 4 RAR␣ and RAR␥ are believed to be most important in the regulation of granulopoiesis. 4,9 This is most strikingly demonstrated in progenitor assays of bone marrow cells derived from RAR␣1 Ϫ/Ϫ RAR␥ Ϫ/Ϫ mice. These cells displayed a block in granulopoiesis at the myelocyte stage of development, revealing an absolute requirement for RAR-mediated signaling in the terminal differentiation of granulocytes. 9 Our understanding of the mechanisms through which RAR␣ mediates cell cycle arrest and differentiation of myeloid cells is a question of particular relevance. First, RAR␣, but not RAR␤ or RAR␥, is involved in the pathogenesis of myeloid leukemia. 10 RAR␣ is involved in leukemia both as a fusion partner, most commonly involving promyelocytic leukemia...

The role of vitamin A and retinoic acid receptor signaling in post-natal maintenance of bone

“…These two studies used different compounds to selectively target RARα and RARγ. A direct comparison of RARα agonists IRX5183 and GR104 revealed IRX5183 has a higher binding affinity and selective activation than GR104 (80,81). The difference in receptor subtype selectivity could explain the differences between these studies.…”

Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis

“…The 5′-MLL fragment covering MLL exons 1-7 (a kind gift from H. Hirai, The University of Tokyo), and the full length of SEPT6 type I (16), were inserted into pMXs-neo to generate pMXs-neo-5′-MLL and pMXs-neo-SEPT6, respectively. Several fragments containing portions of SEPT6, ENL (a kind gift from M. Seto, Aichi Cancer Center Research Institute, Aichi, Japan), or the mutant LBD of hER (29), produced with PCR, were cloned into pMXs-neo-5′-MLL for various pMXs-neo constructs. Each fragment inserted into pMXs-neo was fused with a FLAG or HA epitope tag at the C-terminus.…”

Transcriptional control of granulocyte and monocyte development

“…Regulation of the C/EBPe promoter by RARa may account in part for the role of RARs in granulopoiesis . RARs also activate the CD18 promoter in maturing myeloid cells, in cooperation with the Ets family member GA-binding protein, and mediate their cell cycle arrest (Bush et al, 2003;Walkley et al, 2004). Retinoic acid increases the formation of CFU-G at the expense of CFU-M from marrow progenitors, and 1a,25-dihydroxyvitamin D 3 , which activates the related vitamin D receptor (VDR):RXR complex, increases CFU-M at the expense of CFU-G (Douer and Koeffler, 1982;Koeffler et al, 1984).…”