Direct Supervisor: Francesco Blasi, MD
Location: Unit of Molecular Genetics, Department of Functional Genomics
Descriptive title of the research activity: Role of uPAR in hematopoietic stem cell mobilization
Overall goals: We have recently established a parallelism between Hematopoietic Stem Cells (HSC) Mobilization and the expression of uPAR in human healthy volunteers. Moreover we have preliminary data in collaboration demonstrating a defect in HSC mobilization in uPAR Ko mice. The aim of the work is to dissect the mechanisms underlying this defect.
Rationale and Significance: UPAR has been known to have chemotactic activity mediated by interaction with G protein coupled receptors and integrins, and to affect integrins function and hence signaling and cell adhesion. The defect in uPAR KO mice might be due to a defective mobilization or homing, or both. In particular, we have shown that the cleaved uPAR (D2D3) has chemotactic activity on a CD34+ cell line, mimicking an HSC, and desensitizes these cells as well as CD34+ primary cells to the action of SDF-1. Therefore it is relevant to investigate the state of activity of integrins and the response to D2D3 of CD34+ cells in vitro, as well as the functions of D2D3 on HSC mobilization in vivo.
Description of work: We will employ both in vitro and in vivo methods, and both a mouse and a human system. As a “human” system we will employ a scid-hu (or similar) mice, in which the mice have been transplanted. On these mice, we will test the response of the mice to the administration of D2D3 (alone or in combination with registered mobilization schemes, i.e. 5-FU treatment of G-CSF). Moreover, we will obtain CD34 + cells from healthy volunteers and we will study the response of these cells to D2D3 testing the chemotactic activity, the adhesion properties and the state of activation of integrin-dependent signaling molecules, in particular those known to depend on uPAR. We will also employ the uPAR KO mouse and will test the role of the stroma and of the bone marrow in the deficient HSC mobilization, by transplanting lethally irradiated wt or uPARKO recipients with bone marrow from uPAR or wt dononors, and measuring the repopulation activity, and the mobilization.
Methodology: Main methodologies: A) setting up of the Scid-hu mice. For this we have expertise in house (Dr. Alessandro Aiuti). Dr. Aiuti has published several papers on this system (f.e. Ficara et al., Il-3 or Il-7 favor the maintenance of ADA-scid lymphoid progenitors during ex-vivo gene transfer Molecular Therapy, 2004, 10:1096-108). B) Direct assessment of molecular interactions by advanced fluorescence microscopy (FCS, Fluorescence Correlation Spectroscopy; FRET, FLIM, confocal analysis, etc.). C) Biochemical identification of interacting protein using the TAP (Tandem Affinity Purification) technology coupled to Mass Spectrometry Sequencing Analysis.