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Biomedical subjects

Louise E Purton

Publications and source records attributed to Louise E Purton.

9 recordsLinked to original sources
PubMed2006

RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation.

Hematopoietic stem cells (HSCs) sustain lifelong production of all blood cell types through finely balanced divisions leading to self-renewal and differentiation. Although several genes influencing HSC self-renewal have been identified, to date no gene has been described that, when activated, enhances HSC self-renewal and, when inactivated [corrected] promotes HSC differentiation. We observe that the retinoic acid receptor (RAR)gamma is selectively expressed in primitive hematopoietic precursors and that the bone marrow of RARgamma knockout mice exhibit markedly reduced numbers of HSCs associated with increased numbers of more mature progenitor cells compared with wild-type mice. In contrast, RARalpha is widely expressed in hematopoietic cells, but RARalpha knockout mice do not exhibit any HSC or progenitor abnormalities. Primitive hematopoietic precursors overexpressing RARalpha differentiate predominantly to granulocytes in short-term culture, whereas those overexpressing RARgamma exhibit a much more undifferentiated phenotype. Furthermore, loss of RARgamma abrogated the potentiating effects of all-trans retinoic acid on the maintenance of HSCs in ex vivo culture. Finally, pharmacological activation of RARgamma ex vivo promotes HSC self-renewal, as demonstrated by serial transplant studies. We conclude that the RARs have distinct roles in hematopoiesis and that RARgamma is a critical physiological and pharmacological regulator of the balance between HSC self-renewal and differentiation.

Animals↗
PubMed2005

Cell division and hematopoietic stem cells: not always exhausting.

Pluripotent hematopoietic stem cells (HSCs) sustain blood cell production throughout an individual's lifespan through complex processes supported by self-renewal, differentiation, senescence or cell death decisions of the HSCs. These decisions are tightly regulated under homeostatic conditions, allowing both the continuous generation of progenitors and mature cells in addition to the maintenance and replenishment of the HSC pool. Several recent studies have provided insights into some of the key molecular mechanisms regulating these different cell fate decisions. One of the emerging themes of these studies is that of the importance of cell cycle regulators in the maintenance of HSCs.

Animals↗
PubMed2005

Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are capable of shifting from a state of relative quiescence under homeostatic conditions to rapid proliferation under conditions of stress. The mechanisms that regulate the relative quiescence of stem cells and its association with self-renewal are unclear, as is the contribution of molecular regulators of the cell cycle to these decisions. Understanding the mechanisms that govern these transitions will provide important insights into cell-cycle regulation of HSCs and possible therapeutic approaches to expand HSCs. We have investigated the role of two negative regulators of the cell cycle, p27(Kip1) and MAD1, in controlling this transition. Here we show that Mad1(-/-)p27(Kip1-/-) bone marrow has a 5.7-fold increase in the frequency of stem cells, and surprisingly, an expanded pool of quiescent HSCs. However, Mad1(-/-)p27(Kip1-/-) stem cells exhibit an enhanced proliferative response under conditions of stress, such as cytokine stimulation in vitro and regeneration of the haematopoietic system after ablation in vivo. Together these data demonstrate that the MYC-antagonist MAD1 and cyclin-dependent kinase inhibitor p27(Kip1) cooperate to regulate the self-renewal and differentiation of HSCs in a context-dependent manner.

Animals↗
PubMed2005

Contrasting effects of P-selectin and E-selectin on the differentiation of murine hematopoietic progenitor cells.

OBJECTIVE: The two endothelial selectins, P- and E-selectin, are critically important for adhesion and homing of hematopoietic progenitor cells (HPC) into the bone marrow. Little is known, however, about the roles of these two selectins in hematopoiesis. Here, we demonstrate that the most primitive HPC capable of long-term in vivo repopulation express P-selectin glycoprotein ligand-1/CD162 (PSGL-1), a receptor common to both P- and E-selectin. In addition, we demonstrate that P-selectin delays the differentiation of HPC whereas E-selectin enhances their differentiation along the monocyte/granulocyte pathway, describing different roles for these selectins in the regulation of hematopoiesis. MATERIALS AND METHODS: Murine bone marrow HPC were isolated according to their expression of c-kit and PSGL-1, transplanted into lethally irradiated congenic recipients, and chimerism analyzed 6 months posttransplant. Bone marrow lineage-negative (Lin(-)) Sca-1(+)c-kit(+) cells were then cultured on immobilized P- or E-selectin for 4 weeks in the presence of cytokines. Hematopoietic potential was assessed using in vitro phenotyping and colony-forming assays and in vivo spleen colony-forming unit (CFU-S) and long-term competitive repopulation assays. RESULTS: Long-term competitive repopulating HSCs were Lin(-)c-kit(bright) and expressed intermediate levels of PSGL-1. Both P- and E-selectin slowed the proliferation of Lin(-)Sca-1(+)c-kit(+) cells during the first two weeks of liquid culture. After two weeks, however, cells cultured on immobilized P-selectin showed increased proliferation with increased production of both colony-forming cells (CFC) and CFU-S(12) compared to the other cultures. In contrast, E-selectin enhanced the differentiation of Lin(-)Sca-1(+)c-kit(+) cells into cells that expressed the granulocyte maturation marker, Gr-1, accompanied by loss of CFC potential from these cultured cells. Finally, the long-term repopulation potential of these cells was not maintained following culture on either selectin. CONCLUSION: These results suggest that the two endothelial selectins, E-selectin and P-selectin, have very different effects on HPC. E-selectin accelerates the differentiation of maturing HPC towards granulocyte and monocyte lineages while maintaining the production of more immature CFU-S(12) in ex vivo liquid suspension culture. In marked contrast, P-selectin delays the differentiation of Lin(-)Sca-1(+)c-kit(+) cells, allowing enhanced ex vivo expansion of CFC and CFU-S(12) but not HSCs.

Animals↗
PubMed2004

Osteopenia in Siah1a mutant mice.

Siah1a has been implicated in numerous signaling pathways because of its ability to induce ubiquitin-mediated degradation of many protein substrates. Siah1a knockout mice are growth-retarded, exhibit early lethality, and display spermatogenic defects. In this study we identified a striking low bone volume phenotype in these mice (trabecular bone volume was halved compared with wild type mice), linking Siah1a to bone metabolism for the first time. Markers of bone formation, including osteoblast numbers and osteoid volume, were decreased by up to 40%, whereas the number of osteoclasts was more than doubled in Siah1a mutant mice. However, ex vivo osteoclast formation occurs normally and hematopoietic osteoclast progenitor cell types were present in normal numbers in Siah1a mutant mice. Moreover, adoptive transfer of Siah1a mutant bone marrow into wild type mice failed to reproduce the osteopenia or increased osteoclast numbers observed in mutant mice. Although ex vivo osteoblast colony formation was normal in Siah1a mutant mice, mineralization from these cells was elevated in cultures from Siah1a mutant mice, which may explain the reduction in osteoid volume seen in vivo. These findings suggest that although Siah1a is clearly essential for normal bone metabolism, the bone defect in Siah1a mutant mice is not due to cell-autonomous requirements for Siah1a in osteoblast or osteoclast formation. We propose that bone metabolism defects in Siah1a mutant mice are secondary to an alteration in an unidentified systemic, paracrine, or metabolic factor in these mice.

Adoptive Transfer↗
PubMed2003

Identification of the molecular requirements for an RAR alpha-mediated cell cycle arrest during granulocytic differentiation.

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 retinoic acid receptor alpha (RAR alpha), but not RAR beta or gamma, required both Mad1 and p27(Kip1) to induce cell cycle arrest and to accelerate terminal differentiation of granulocytes. Although RAR alpha did not directly regulate Mad1 or p27(Kip1), the RAR alpha target gene C/EBP epsilon directly regulated transcription of Mad1. Induction of C/EBP epsilon activity in granulocytic cells led to rapid induction of Mad1 protein and transcript, with direct binding of C/EBP epsilon to the Mad1 promoter demonstrated through chromatin immunoprecipitation assay. These data demonstrate that cell cycle arrest in response to RAR alpha specifically requires Mad1 and p27(Kip1) and that Mad1 is transcriptionally activated by CCAAT/enhancer-binding protein epsilon (C/EBP epsilon). Moreover, these data demonstrate selectivity among the RARs for cell cycle arrest pathways and provide a direct mechanism to link differentiation induction and regulation of the Myc antagonist Mad1.

Animals↗
PubMed2003

Generation and analysis of Siah2 mutant mice.

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.

Animals↗
PubMed2002

An oxysterol-binding protein family identified in the mouse.

Oxysterols are oxygenated derivatives of cholesterol. They have been shown to influence a variety of biological functions including sterol metabolism, lipid trafficking, and apoptosis. Recently, 12 human OSBP-related genes have been identified. In this study, we have identified a family of 12 oxysterol-binding protein (OSBP)-related proteins (ORPs) in the mouse. A high level of amino acid identity (88-97%) was determined between mouse and human ORPs, indicating a very high degree of evolutionary conservation. All proteins identified contained the conserved OSBP amino acid sequence signature motif "EQVSHHPP," and most contained a pleckstrin homology (PH) domain. Using RT-PCR, each mouse ORP gene was found to exhibit a unique tissue distribution with many showing high expression in testicular, brain, and heart tissues. Interestingly, the tissue distribution of ORP-4 and ORP-10 were the most selective within the family. Expression of the various ORP genes was also investigated, specifically in highly purified populations of hemopoietic precursor cells defined by the lin(-) c-kit(+) Sca-1(+) (LKS(+)) and lin(-) c-kit(+) Sca-1(-) (LKS(-)) immunophenotype. Most ORP genes were expressed in both LKS(+) and LKS(-) populations, although ORP-4 appeared to be more highly expressed in the primitive, stem-cell enriched LKS(+) population, whereas ORP-10 was more highly expressed by maturing LKS(-) cells. The identification of a family of ORP proteins in the mouse, the frequently preferred animal model for in vivo studies, should further our understanding of the function of these proteins and their interactions with each other.

Amino Acid Sequence↗