Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6)

Abstract

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo͞Ark), Sky (Dtk͞Tyro3͞Rse͞ Brt͞Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively ␥-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be ␥-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells.

Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase

“…Chen et al (1997) have described interactions of the ligand Gas6 and the Mer/Axl/ Tyro-3 family of RTKs as being transforming in vitro (Chen et al, 1997) and Dirks et al (1999) have found these interactions to be associated with leukemia and lymphoma cell line proliferation. The Gas6-mediated stimulation of Mer that we observed in vitro should also occur with the Mer Tg lymphocytes in the bone marrow and peripheral blood in vivo, as Gas6 is produced by bone marrow stromal cells (Dormady et al, 2000) and is found in the plasma (Borgel et al, 2006).…”

Axl, a prognostic and therapeutic target in acute myeloid leukemia mediates paracrine crosstalk of leukemia cells with bone marrow stroma

“…Subsequently, we added human IL-10 and hM-CSF function-blocking antibodies to the cocultures and found that the presence of both anti-hIL-10 and antihM-CSF attenuated mGas6 upregulation, whereas each antibody alone had no effect ( Figure 1H). Interestingly, this stroma-derived Gas6 was partially responsible for the well-known protective effect of BMDSCs on AML cells [34][35][36] because the pro-survival effect of S17 and OP9 cells on HL60 cells was reduced by sAxl, a Gas6-neutralizing "trap" molecule (supplemental Figure 1D). 37 In addition, we found that Gas6 contributed to the chemoprotective effect of BMDSCs on AML cells because sAxl decreased their pro-survival effect upon treatment with cytarabine ( Figure 1I).…”

GAS6 is an estrogen-inducible gene in mammary epithelial cells

“…X59846) attracted our special attention. As a secreted protein, GAS6 plays an important role in sustaining hematopoietic progenitor cells growth (25) and can induce mesangial cell proliferation (26). GAS6 could be involved in mediating the action of estrogen in mammary glands.…”

“…Although amphiregulin is induced by estrogen and can stimulate the proliferation of mammary epithelial cells (27), mammary glands from the mouse model with amphiregulin null mutation can still respond to estrogen (28), indicating that there are other factors mediating the action of estrogen. Given the role of GAS6 in stimulating the proliferation of other cells (25,26) and its induction in mammary glands by estrogen, GAS6 could be one of the factors involved in the estrogen-stimulated ductal growth of mammary glands. Enhancement of GAS6 mRNA expression in normal mammary epithelial cells and ER positive breast cancer cells by estrogen.…”