Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Effects of fenugreek (Trigonella foenum graecum) on gilthead seabream (Sparus aurata L.) immune status and growth performance.

The possible effect of dietary administration of fenugreek (Trigonella foenum graecum) on gilthead seabream (Sparus aurata L.) immune status and growth performance was studied. Fish were divided into 4 groups before being fed with commercial diet supplemented with 0% (control), 1%, 5% and 10% of fenugreek seeds for 4 weeks. The effects of the diets were analysed on the cellular (respiratory burst activity and leucocyte peroxidase content) and humoral (complement activity, antiprotease, total protein, peroxidase, and IgM level) immune parameters, as well as growth and haematological parameters (WBC and RBC counts). The results recorded enhancement in all the assayed parameters in fish fed fenugreek diets comparing to control fish. The expression of several immune-related genes in head-kidney (MHC1, CSF-1R, IL-8, and IgM) and different antioxidant enzyme genes in liver (GR, CAT and SOD) of seabream specimens were also investigated. Again, the highest fenugreek doses tested provoked significant up-regulation in most of immune-related genes and antioxidant enzyme genes (p < 0.05). No adverse effects were observed on intestine and liver morphology on fish fed fenugreek diets. The present results suggest that the fenugreek seed, specially the highest dosage used in the present work could be considered a good food supplement to improve the immune status and increase the production of gilthead seabream.

The Ig-like domain of human GM-CSF receptor alpha plays a critical role in cytokine binding and receptor activation.

GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.

Hydrogen peroxide generated extracellularly by receptor-ligand interaction facilitates cell signaling.

Reactive oxygen species (ROS) are key components of postreceptor intracellular signaling pathways; however, the role of ROS in signal initiation is uncertain. We discovered that receptor-ligand interaction caused the generation of hydrogen peroxide (H2O2). Using members of the hematopoietin receptor superfamily, as well as EGF receptor, we show that H2O2 is generated by specific receptor-ligand interaction in cells and in cell-free systems. With cognate ligand, the extracellular domain of the receptor was sufficient for H2O2 generation. We also found that production of H2O2 was diminished in a granulocyte-macrophage colony-stimulating factor receptor mutant unable to bind ligand. Exogenously added H2O2 induced signaling in the absence of ligand, whereas catalase and a membrane-bound peroxiredoxin inhibited ligand-dependent signaling. Our results suggest that H2O2 produced by receptor-ligand interaction is involved as a chemical mediator that facilitates cell signaling.

Identification of conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit critical for function. Evidence for formation of a heterodimeric receptor complex prior to ligand binding.

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues, a tryptophan-serine motif (WSXWS) in the extracellular domain, and a proline-rich cytoplasmic domain. The high affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (hGM-CSFR) consists of two subunits, alpha (hGM-CSFR alpha) and beta (hGM-CSFR beta), both of which are members of the receptor superfamily. In this study, we prepared mutations in conserved amino acids of the receptor subunit necessary for GM-CSF binding (hGM-CSFR alpha) and analyzed mutant receptors for low affinity binding, internalization, and high affinity binding when complexed with the beta subunit. Mutations in the cytoplasmic domain did not affect GM-CSF binding or receptor internalization. Mutation of a single conserved serine residue within the WSXWS motif diminishes cell surface receptor expression but not ligand binding. Mutation of either the second or third conserved cysteine residue of hGM-CSFR alpha resulted in complete loss of low affinity binding; however, co-expression of the cysteine 2 mutant with hGM-CSFR beta yielded a high affinity receptor complex. Since neither the cysteine 2 mutant nor the beta subunit can bind ligand alone, this result suggests that hGM-CSFR alpha and hGM-CSFR beta exist in a preformed heterodimeric protein complex on the plasma membrane.