Hematopoiesis in mammals undergoes a developmental shift in location from fetal liver to bone marrow accompanied by a gradual transition from highly proliferative to deeply quiescent stem cell populations. P2Y receptors are G-protein-coupled nucleotide receptors participating in vascular and immune responses to injury. We identified a P2Y-like receptor for UDP-conjugated sugars, GPR105 (P2Y 14 ), with restricted expression on primitive cells in the hematopoietic lineage. Anti-GPR105 antibody selectively isolated a subset of hematopoietic cells within the fetal bone marrow, but not in the fetal liver, that was enriched for G0 cell cycle status and for in vitro stem-cell-like multipotential long-term culture capability. Conditioned media from bone marrow stroma induced receptor activation and chemotaxis that was sensitive to G␣i and anti-receptor antibody inhibition. GPR105 is a G-protein-coupled receptor identifying a quiescent, primitive population of hematopoietic cells restricted to bone marrow. It mediates primitive cell responses to specific hematopoietic microenvironments and extends the known immune system functions of P2Y receptors to the stem cell level. These data suggest a new class of receptors participating in the regulation of the stem cell compartment. G-protein-coupled receptors (GPRs) have a broad repertoire of activating ligands ranging from photons to chemokines and induce an array of cellular events in virtually every physiologic system. Yet there remains a large proportion of GPR without known ligands or with limited known functions. A group of GPRs responsive to nucleotides (termed P2Y receptors) has been defined mediating cell-cell communication in the nervous system and in modulating vascular tone . A well-defined effect of nucleotides on platelet activation and vascular smooth muscle migration and growth has suggested participation of P2Y receptors in the response to injury. More recently, these receptors have been noted to affect cellular constituents of the innate immune system altering functional characteristics of monocytes, eosinophils, and dendritic cells and to play a critical role in terminating the inflammatory response in vivo (Mutini et al. 1999;Ferrari et al. 2000;Idzko et al. 2001;Santiago-Perez et al. 2001;Warny et al. 2001;Wilkin et al. 2001). The P2Y receptor specificity originally thought to be restricted to purine (adenine) nucleotides has been extended to pyrimidine nucleotides (uridine) and more recently to a receptor with specificity for UDP, but only when conjugated to glucose or related sugars (Chambers et al. 2000). This receptor, GPR105 (recently designated P2Y 14 ; Abbracchio et al. 2003), was originally noted to be expressed in rat brain tegmentum, but has no known function apart from being the presumed basis for UDP-glucose to induce diaphragmatic contraction or neural action potentials (Pastoris et al. 1979(Pastoris et al. , 1981. We provide evidence that this receptor participates in regulation of hematopoietic cells with stem cell characteristics.

Summary A recent surge in obesity has given impetus to better understand the mechanisms of adipogenesis, particularly brown adipose tissue (BAT) due to its potential utilization for anti-obesity therapy. Postnatal brown adipocytes arise from early muscle-progenitors but how brown fat lineage is determined is not completely understood. Here, we show that a multifunctional protein EWS (Ewing Sarcoma) is essential for determining brown fat lineage during development. BATs from Ews-null embryos and newborns are developmentally arrested. Ews mutant brown preadipocytes fail to differentiate due to loss of Bmp7 expression, a critical early brown adipogenic factor. We demonstrate that EWS, along with its binding partner YBX1 (Y-box binding protein 1), activates Bmp7 transcription. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Remarkably, Ews-null BATs and brown preadipocytes ectopically express myogenic genes. These results demonstrate that EWS is essential for early brown fat lineage determination.

A stromal protein, designated restrictin-P, that specifically kills plasma-like cells was purified to homogeneity and shown to be identical with activin A. The specificity to plasma-like cells stemmed from the ability of restrictin-P/activin A to competitively antagonize the proliferation-inducing effects of interleukin (IL) 6 and IL-11. Restrictin-P further interfered with the IL-6-induced secretion of acute phase proteins by HepG2 human hepatoma cells and with the IL-6-mediated differentiation of M1 myeloblasts. A competition binding assay indicated that restrictin-P did not interfere with the binding of IL-6 to its receptor on plasma-like cells, suggesting that it may act by intervening in the signal transduction pathway of the growth factor. Indeed, concomitant addition of restrictin-P and IL-6 to cytokinedeprived B9 hybridoma cells was followed by sustained overexpression of junB gene until cell death occurred, while IL-6 alone caused a transient increase only. This altered response to IL-6 stimulation was accompanied by a moderate increase in STAT protein activation. Thus, in this study, we identified the plasmacytoma growth inhibitor, restrictin-P, as being activin A of stromal origin. It is shown that activin A is an antagonist of IL-6-induced functions and that it modifies the IL-6 signaling pattern.

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Hematopoietic stem progenitor cells (HSPCs) are present in very small numbers in the circulating blood in steady-state conditions. In response to stress or injury, HSPCs are primed to migrate out of their niche to peripheral blood. Mobilized HSPCs are now commonly used as stem cell sources due to faster engraftment and reduced risk of posttransplant infection. In this study, we demonstrated that a nucleotide sugar, UDP-glucose, which is released into extracellular fluids in response to stress, mediates HSPC mobilization. UDP-glucosemobilized cells possessed the capacity to achieve long-term repopulation in lethally irradiated animals and the ability to differentiate into multi-lineage blood cells. Compared with G-CSF-mobilized cells, UDP-glucose-mobilized cells preferentially supported long-term repopulation and exhibited lymphoid-biased differentiation, suggesting that UDP-glucose triggers the mobilization of functionally distinct subsets of HSPCs. Furthermore, co-administration of UDP-glucose and G-CSF led to greater HSPC mobilization than G-CSF alone. Administration of the antioxidant agent NAC significantly reduced UDP-glucose-induced mobilization, coinciding with a reduction in RANKL and osteoclastogenesis. These findings provide direct evidence demonstrating a potential role for UDP-glucose in HSPC mobilization and may provide an attractive strategy to improve the yield of stem cells in poor-mobilizing allogeneic or autologous donors.