Abstract. Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial ceils. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU-GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34 ÷ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.

Granulocyte macrophage colony-stimulating factor (GM-CSF) has emerged as an important growth factor for trophoblast and other placental cells, leading to improved placental functioning and fetal survival. Recent observations have indicated that GM-CSF is synthesized by epithelial cells in the murine pregnant and non-pregnant uterus. In this study, the production of GM-CSF by cells derived from human endometrium is assessed using a sensitive bioassay and specific neutralization of the cytokine bioactivity with a monoclonal antibody to GM-CSF. Originally, GM-CSF was assayed in the culture supernatants of explant cultures of human endometria. Concentrations of GM-CSF up to 4440 pg/ml were detected. Subsequently, enriched epithelial cell cultures were prepared from glands isolated from human endometrium. The purity of epithelial cultures was demonstrated by the expression of cytokeratin, a weak immunoreactivity for vimentin and a lack of immunoreactivity for leukocyte common antigen, CD68, a macrophage-specific protein and endothelial marker (factor VIII-related antigens). Detected concentrations of GM-CSF were as high as 18,800 pg/ml. Furthermore, pure epithelial cells of a neoplastic endometrial cell line ECC1 secreted GM-CSF, confirming the ability of endometrial epithelial cells to secrete this cytokine. The immunostaining of dated endometria from proliferative and secretory phases showed primarily that epithelial cells, and to a lesser extent stromal cells, exhibited immunoreactivity for GM-CSF. A Western blot analysis, performed to validate the immunohistochemical data, confirmed the presence of an immunoreactive gene product for GM-CSF in human endometrium throughout the menstrual cycle. These findings indicate that human endometrium synthesizes GM-CSF and that epithelial cells are a major contributor to its production.

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients' plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.

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Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte- conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.