M Siena

Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.

Physiological levels of 1α, 25 dihydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors

Although supraphysiological levels of 1α, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo‐ and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid‐culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38− hematopoietic stem cells, the down‐regulation of CD34 antigen, and the increase of monocyte‐committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.

The Kinetic Status of Hematopoietic Stem Cell Subpopulations Underlies a Differential Expression of Genes Involved in Self‐Renewal, Commitment, and Engraftment

The gene expression profile of CD34− hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage‐negative (Lin−) CD34−, Lin−CD34+, and Lin+CD34+ cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34− cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin‐dependent kinases (CDKs), CDK inhibitors, and growth‐arrest genes. According to the expression of their receptors and transducers, interleukin (IL)‐10 and IL‐17 showed an inhibitory effect on the clonogenic activity of CD34− cells. Conversely, CD34+ cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34− cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self‐renewal and lineage commitment. The preferential expression in CD34+ cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34+ cells. Thus, the different kinetic status of CD34− and CD34+ cells, detailed by molecular and functional analysis, significantly influences their biological behavior.

Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14+ human normal myeloid precursors

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14− and CD14+ myeloid precursors generated by ‘in vitro’ culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14− cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.

Gene expression profile of Vitamin D3 treated HL60 cells shows an incomplete molecular phenotypic conversion to monocytes.

By high density oligonucleotide microarrays we have studied the expression profile of proliferating and VD treated HL60 cells and the molecular phenotype of VD monocytes and that of CD14+ peripheral monocytes has been compared. The results indicate that important changes in functional categories of the differentially expressed genes underlie the differentiation transition from myeloblasts to monocytes. This differential gene expression pattern leads to an increased expression of mRNAs involved in surface and external activities since many of the VD induced genes belong to ligand binding, receptors, cell surface antigens, defense/immunity and adhesion molecules functional categories. The results also indicate that the molecular phenotypes of monocytes and VD induced cells diverge for a small but significant set of defense related genes. Particularly, class II MHC genes are not expressed in these cells. Furthermore, the high levels of expression of these genes induced by serum treatment of monocytes are decreased by VD.

Requirement of the coiled-coil domains of p92 c-Fes for nuclear localization in myeloid cells upon induction of differentiation

The nonreceptor tyrosine kinase Fes is implicated in myeloid cells differentiation. It has been observed that its localization can be cytoplasmic, perinuclear, or nuclear. To further characterize this point, we studied Fes subcellular localization in myeloid cell lines (HL60 and K562) and in COS1 cells. Fes was observed in both the nucleus and the cytoplasm of HL60, K562 cells overexpressing Fes and only in the cytoplasm of COS1 cells, suggesting that nuclear localization is cell context dependent. Moreover, in myeloid cells, the treatment with differentiation-inducing agents such as retinoic acid, phorbol esters and vitamin D, is followed by an increase of the oligomeric form of Fes in the nucleus. In fact, oligomerization seems to be necessary for translocation to occur, since Fes mutants missing the coiled-coil domains are not able to form oligomers and fail to localize in the nucleus. The active form of Fes is tyrosine phosphorylated; however, phosphorylation is not required for Fes to localize in the nucleus, since tyrosine kinase inhibitors do not block the translocation process.

Development of an IL-6 antagonist peptide that induces apoptosis in 7TD1 cells

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.