Expression and Functionality of the zyxwv trkA Proto-Oncogene Product/NGF Receptor in Undifferentiated Hematopoietic Cells zy By zyxwvutsrqponm Sylvie Chevalier, Vincent Praloran, Carthage Smith, Dona1 Mac Grogan, Nancy Y. Ip, George D. Yancopoulos, Philippe Brachet, Annick Pouplard, and Hugues Gascan The expression of the low-affinity NGF receptor (p75) and the trkA proto-oncogene product was analyzed in a series of human hematopoietic cell lines at protein and RNA lev- els. We did not detect any zyxwvutsrq form of NGF receptor in cell lines displaying a myelomonocytic phenotype (HL60 and U937). In contrast, cells displaying a more immature erythroleu- kemic phenotype (TFI and K562) expressed TrkA in the absence of detectable p75. Scatchard analysis showed a single high-affinity site for NGF /kd = 10"" mol/L), with a copy number ranging from 300 to 3,000 sites per cell de- pending on the studied cell line.In addition, NGF induced autophosphorylation of TrkA and could substitute for gran- ulocyte-monocyte colony-stimulating factor to trigger the HE PROLIFERATION and the differentiation of he- matopoietic stem cells is regulated by a series of poly- peptide growth factors referred to as colony-stimulating fac- tors (CSFs) and interleukins (ILs).' The successive action of these mediators on bone marrow precursor cells allows for their maturation into functional blood cells. Recent studies have characterized the receptors of numerous cytokines in- volved in these processes and defined a family of molecules defined as cytokine receptors, which share some conserved motifs' as well zyxwvutsr as having functional ~imilarities.~ Besides the cytokine receptor family, receptor tyrosine kinases encoded by thefms proto-oncogene (macrophage-CSF [M-CSF] re- cepto~)~ and kit proto-oncogene (stem cell factor receptor)5 also transduce important signals for hematopoietic cell mat- uration. In addition to the classical CSFs and ILs, some other cy- tokines also display discrete hematopoietic stimulating ac- tivities. This is the case for NGF, classically known for its activity on the survival, development, and maintenance of embryonic peripheral sympathetic and sensory neurons, and also identified as a neurotrophic factorfor some central neurons.6 More recently, it has been shown that injections of NGF into neonatal rats result in a significant increase of tissue mast cell number, and that NGF promotes mainly eosinophil and basophil/mast cell colony formation in semisolid medium culture conditions in vitro.7" I Human and rat NGF receptors were initially cloned, with the help of monoclonal antibodies (MoAbs) recognizing a 75-kD protein (hereon p75) expressed on the surface of neu- ral crest-derived cells.12.13 However, transfection in eukary- otic cells of plasmids encoding this molecule led to theex- pression of putative receptors displaying only a low affinity for NGF, with a dissociation constant in the range of lo-* to IO-' mol/L. In addition to p75, another molecule identi- fied as the zyxwvutsrqpo trk proto-oncogene product also binds NGF,'"l6 The trk oncogene (tropomyosin receptor kinase) was origi- nally identified as a transforming gene from a human colon carcinoma, formed by a fusion between tropomyosin and a tyrosine kinase.",'* trkB and ~rkC,~'-" genes closely related to trk (hereon trkA), encode protein tyrosine kinases that serve as receptors for the NGF-related ligands: brain&- T zyxw Blood. Vol83. No 6 (March 15). 1994: pp 1479-1485 proliferation of the TFI cell line, with a half-maximal signal observed at 50 pmol/L, indicating that p75 is not required for DNA synthesis in this cell line. The physiologic rele- vance of NGF in early hematopoiesis was confirmedby showing that 12% to 15% of progenitor blood cells from mice treated with 5-fluorouracil expressed TrkA and that these cells could be induced to proliferate and differentiate in responseto NGF in association with macrophage colony- stimulating factor. Our study demonstrates for the first time that rrkA proto-oncogene expression and activation is not restricted to the nervous system,but is also an impor- tant element in early hematopoiesis. zyx 0 1994 by The American Society of Hematology. rived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4).25-2y Binding of NGF to TrkA induces its autophosphorylation as well as subsequent sig- naling events inside the cell. Binding studies for NGF and TrkA have produced conflicting results depending on the investigators. In some experiments, TrkA by itself displayed a high-affinity binding site for NGF (10"' to lo-" mol/L) and was sufficient for cellular signal transd~ction.'~ In other systems, TrkA, as p75, only exhibited a low affinity for the ligand, and a coexpression of both TrkA and p75 was re- quired for the formation of a functional receptor with a high affinity for NGF.~~ The differentiation and proliferation of progenitors from bone marrow to mature mast cells is driven by several cy- tokines belonging to the CSF andIL family. This property is shared by IL-3, IL-4, IL-9, granulocyte-macrophage CSF (GM-CSF), and stem cell factor.30 A puzzling correlation is the ability of the same growth factors to also behave as costimulating activities in hematopoietic stem cell assays, suggesting a direct relationship between hematopoietic stem cells and mature mast cells.30 Several reports have demon- strated the ability of NGF to act as a mast cell growth factor alone or in combination with GM-CSF."" These observa- tions prompted us to analyze the effects of NGF on un- differentiated myeloid progenitors by using in vitro estab- lished tumor cell lines or stem cell primary cultures. From the Neurobiologie et immunopathologie des maladies du systime nerveux, INSERM U 298, CHR U Angers, Angers, France; the Laboratoire d'Himatologie, CHR U Limoges, Limoges, France; and Regeneron Pharmaceuticals, Inc, Tarrytown, NY. Submitted July I, 1993; accepted October 28, 1993. Supporled by a grant ofAssociation pour la Recherche sur le Can- cer (Villejuif;France). Address reprint requests to Hugues Gascan, PhD, INSERM U 298, CHRU, 49033 Angers Cedex zyxw 01, France. The publication costs of this article were defrayed in part by page charge payment.This article must therejore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solelyto indicate thisfact. 0 1994 by The American Society of Hematology. 0006-4971/94/8306-0010$3.00/0 1479 Downloaded from http://ashpublications.org/blood/article-pdf/83/6/1479/612755/1479.pdf by guest on 16 August 2022