Gi-Coupled GPCR Signaling Is Required at Multiple Levels and In Different Lineages In Hematopoiesis.

Abstract 2635

G protein-coupled receptors (GPCRs) can regulate cell migration, survival, proliferation and differentiation –– key processes in hematopoiesis. The Gi-coupled receptor CXCR4 plays a key role in hematopoiesis, suggesting that related receptors might also contribute. Because all Gi family members except Gz are inhibited by pertussis toxin (PTX), we utilized a ROSA26-CreOnPTX mouse line that expresses PTX in a Cre-dependent manner to broadly probe the role of Gi signaling in hematopoiesis. Mice hemizygous for the hematopoietic lineage-specific Cre transgene Vav-iCre were crossed with ROSA26-^{CreOnPTX/CreOnPTX} mice to generate offspring expressing PTX in hematopoietic lineages (Vav-PTX) and Cre-negative controls in which the PTX allele remained silent. Vav-PTX mice were born at the expected Mendelian rate, and except for a smaller thymus, were grossly normal, but all died with pneumonia between days 2 and 14. Bone marrow in 3 day-old Vav-PTX mice was hypocellular with significant underrepresentation of granulocytic and lymphocytic lineages as well as hematopoietic stem and progenitor cells (lin-, c-kit+, Sca1+). In bone marrow reconstitution studies, cells from Vav-PTX fetal livers (E14.5) showed impaired short-term and no long-term repopulating activity. Additionally, Vav-PTX fetal liver cells were significantly impaired in their ability to form granulocyte/macrophage and erythroid colonies in vitro. Interestingly, when wild-type E14.5 fetal liver cells were grown in vitro in presence of exogenous PTX, only erythroid colony formation was impaired, and flow cytometric analysis of the progenitor populations of Vav-PTX fetal liver revealed a significant decrease in granulocyte-macrophage progenitors (GMPs) as well as in common myeloid progenitors (CMPs) but not in megakaryocyte/erythroid progenitors (MEPs). Thus, reduced progenitor populations may account for reduced granulocyte/macrophage colony-forming activity in fetal liver cell cultures but does not account for reduced erythroid colony-forming activity. Indeed, normal MEP numbers in Vav-PTX livers and the ability of exogenous PTX to inhibit formation of erythroid colonies in wild-type fetal liver cultures suggests that Gi signaling in MEPs or their progeny may contribute to erythropoiesis in fetal liver. Several of the necessary roles of Gi signaling identified above are not accounted for by the function of CXCR4, and, taken together, our data suggest that Gi-coupled GPCRs likely contribute to hematopoiesis at multiple levels and in different lineages. An effort to identify GPCRs that contribute to erythropoiesis is underway.