The major objective of this research is to characterize the molecular mechanisms by which granulocyte colony-stimulating factor (G-CSF) acting through its receptor (GCSFR) directs neutrophil production and function. The precise molecular mechanisms by which the G-CSFR mediates these biological responses are largely undefined. To investigate these molecular mechanisms, we have generated a series of mice with targeted (""""""""knock-in"""""""") mutations of their G-CSFR. The first G-CSFR mutation (d715) results in the deletion of nearly half of the cytoplasmic (signaling) domain of the G-CSFR. Surprisingly, mice homozygous for the d715 G-CSFR mutation have normal basal granulopoiesis. A second targeted G-CSFR mutation has been generated in which the sole remaining cytoplasmic tyrosine in the d715 G-CSFR mutant has been changed to phenylalanine. This mutant G-CSFR (d715F) does not activate signal transducer and activator of transcription-3 (STAT-3), the principal STAT protein activated by the G-CSFR. Mice homozygous for the d715F G-CSFR are neutropenic and exhibit a partial block in granulocytic differentiation in the bone marrow. D715F hematopoietic progenitors demonstrate significantly impaired G-CSF dependent cellular proliferation and granulocytic differentiation. The defect in proliferation is due, at least in part, to defect in G-CSF induced survival signals. By characterizing neutrophil production, maturation, and function and G-CSFR signaling cascades in mice homozygous for these G-CSFR mutations, we hope to identify the signal transduction pathways that mediate specific biologic actions of G-CSF. The following specific aims are proposed. 1. We will characterize the effect of the d715F G-CSFR mutation on granulocytic differentiation and G-CSF induced cellular proliferation in targeted transgenic mice. Granulocytic differentiation will be assessed along with the expression of transcription factors known to be involved in granulocytic differentiation. The ability of the d715F G-CSFR to transmit mitogenic signals will be determined. The role of G-CSF dependent activation of STAT-3 on granulocytic differentiation and cellular proliferation will be characterized both in vivo and in vitro. 2. We will characterize the effect of the d715 and d715F G-CSFR mutations on neutrophil function and activation. The functional phenotype of neutrophils isolated from d715 and d715F mice will be characterized in detail. Additionally the in vivo response to infectious agents will be characterized. 3. We will characterize the signal cascades responsible for G-CSF induced survival signals in primary myeloid cells. The effect of the d715F G-CSFR mutation on neutrophil survival in vivo will be assessed. The effect of the d715 and d715F G-CSFR mutations on the activation and expression of certain molecules implicated in survival signaling will be determined. The role of STAT-3 in the transduction of G-CSF induced survival signals will be characterized.
| Souza, Liliana R; Silva, Erica; Calloway, Elissa et al. (2013) G-CSF activation of AKT is not sufficient to prolong neutrophil survival. J Leukoc Biol 93:883-93 |
| Mermel, Craig H; McLemore, Morgan L; Liu, Fulu et al. (2006) Src family kinases are important negative regulators of G-CSF-dependent granulopoiesis. Blood 108:2562-8 |
