Abstract

Hematopoiesis implies a number of different cellular events from self-renewal of stem cells and proliferation to differentiation and migration. All these steps require tight regulation by signaling, and many cytokine signaling pathways have been described. However the signaling mechanisms that govern trafficking, proliferation, survival and differentiation of hematopoietic stem cells and other hematopoietic cell types are poorly understood. G-protein coupled receptors (GPCRs) can regulate many of these processes, and the Gi-coupled receptor CXCR4 is known to play a key role. Roles for other Gi-coupled receptors are just beginning to be uncovered, and given the functions of Gi-signaling in regulation of cell homing, proliferation and survival, it seems likely that Gi-coupled GPCR signaling plays an important role in hematopoiesis. By using a genetic approach – knockout of Gi function in hematopoietic lineages – we are probing the role of Gi-coupled GPCR’s in hematopoiesis to determine which receptors are involved. There are 6 sub-types in the Gi family, and it has been difficult to eliminate all Gi function by gene knockout. However, all Gi family members, except for Gz, are sensitive to inhibition by pertussis toxin (PTX). To ablate Gi signaling in specific cell lineages, we utilized a mouse in which the ROSA26 gene was modified to drive expression of pertussis toxin S1 subunit (PTX) after Cre-mediated excision of a floxed stop cassette. To study the effect of inhibition of Gi signaling in hematopoiesis, we crossed mice carrying one allele of the hematopoietic lineage-specific Cre transgene Vav-iCre with the ROSA26PTX/PTX mice. Half the offspring from this cross carry Vav-Cre Tg+/o: ROSA26PTX/+ (Vav-PTX mice), the other half transgene-negative ROSA26PTX/+ (controls). Vav-PTX mice were born at the expected Mendelian rate. Pups were grossly normal but consistenly displayed a smaller thymus. However, all Vav-PTX mice died between day 2 and 14, presumably from pneumococcal pneumonia. Neonatal Vav-PTX mice showed fewer hematopoietic cells in bone marrow, which was also observed in Vav-PTX embryos collected at E18.5. Thus, defective hematopoiesis was not secondary to postnatal infection or general failure to thrive. Competitive transplantation studies using fetal liver cells from Vav-PTX embryos or control embryos collected at E14.5 showed decreased short-term reconstitution (5weeks) and severely impaired long-term reconstitution (10 and 16 weeks) compared to control fetal liver cells. In contrast, fetal liver cells from CXCR4−/− embryos still showed partial reconstitution after 16 weeks. These data are consistent with defective homing of hematopoietic stem cells (HSCs) from fetal liver to bone marrow, but also with decreased proliferation, survival, and/or differentiation of HSCs once they reach their marrow niche. When fetal liver cells were grown in vitro, cells from Vav-PTX embryos showed decreased survival and proliferation when stimulated with cytokine promoting either B-cell, erythroid or granulocyte/macrophage lineages. This suggested a role for Gi signaling in proliferation in all hematopoietic lineages. In addition, early data suggest that the population of Lin−, c-kit+, Sca1+ cells in fetal liver from Vav-PTX embryos is decreased compared to their littermate controls. Our results suggest a key role for Gi and hence Gi-coupled GPCRs in several mechanisms required for normal hematopoiesis.

Disclosures: Terpager:Lundbeckfonden: Research Funding.

Author notes

Corresponding author