Role Of Tyro3 Receptor Tyrosine Kinases Subfamily In Regulating Of Erythropoiesis In Mice

Axl,Tyro3 and Mer are three members of a recently characterized receptor tyrosinekinase (RTK) family that share a conserved intracellular tyrosine kinase domain and anextracellular domain similar to those seen in cell adhesion molecules.These RTKs bind thevitamin K-dependent protein growth-arrest-specific gene 6 (Gas6),which is structurallyrelated to the protein S anticoagulation factor.Gas6/Tyro3 RTKs signaling regulates cellgrowth and survival.They are widely expressed in various mammalian tissues,such asnervous,immune,reproductive,vascular and hematopoietic tissues,and play an importantrole in regulating spermatogenesis,immune homeostasis,platelet functions,NK cellsdifferentiation,angiogenesis and vascular remodeling.Although they are expressed inhematopoietic system,the role of the three receptors in hematopoiesis is unknown.In thisstudy,we demonstrate that Axl and Mer are critical in regulating erythropoiesis.1.By RT-PCR,Western blot and Immunocytochemistry,we found that Axl and Mer,butnot Tyro3,are expressed in all stages of developing erythroid cells.Marrow stromal cellsexpressed not only all three receptors,but also their common ligands,Gas6 and Protein S.2.We found Axl^-/-Mer^-/- (AM) bone marrows to contain fewer total cells than wild-typecontrols.In contrast,the bone marrow cellularity in single mutant mice (Axl^-/- or Mer^-/-)were not altered significantly compared to wild-type controls.The results suggest that Axland Mer regulate erythropoiesis.3.Anemia was not observed in AM mice.However,we found that the spleens of AM micewere enlarged up to 4 times the wild-type controls.Histological examination showed anevident expansion of red pulps in the spleen.The flow cytometry anlysis showed thepercentage of all stages erythroblasts in AM spleen was increased.The livers of AM miceexpand up to 3.5-fold,and the evident erythropoiesis islands appeared in AM livers.Theresults suggest compensatory extramedullary erythropoiesis occurs in liver and spleen ofAM mice. 4.Although a significant reduction in erythroid cells was observed in AM bone marrows,the number of CFU-E progenitors was increased.However,there was no significantdifference in number of BFU-E progenitors between AM and wild-type bone marrows.Thenumbers of erythroid progenitors in AM bone marrow is twice more than that in wild-typecontrol.However,the numbers of the early,intermediate,late erythroiblasts and mature redcells were significantly reduced in AM bone marrows.By analysis of annexin-V positivecell,we found that apoptosis rate of the different populations of erythroid cells was notsignificantly altered in AM bone marrows and spleens compared to controls.In order toanalyze growth rate of the erythroid progenitors,they were gated and performed akaryotype analysis.The result showed a comparable cell cycle of the erythroid progenitorsin wild-type and AM bone marrows.Taken together,the results suggest that thedifferentiation of erythroid cells is blocked at the transition of the erythroid progenitors toproerythroblasts,which results in the accumulation of erythroid progenitors and thereduction of erythroblasts in AM bone marrow.5.AM mice receiving wild-type bone marrow cells displayed a normal erythropoiesis.Conversely,wild-type mice engrafted with AM bone marrow cells exhibit an impairederythropoiesis,and have a similar distribution of erythroid cells with that in AM mice.Andwild-type mice engrafted with wild-type and AM bone marrow cells also showed a normalerythropoiesis.These results demonstrate that the defect in development of AM erythroidcells is cell autonomous.6.With an effort to reveal the molecular mechanisms underlying the defect oferythropoiesis in AM mice,we found that the expression of GATA-1 and EpoR decline inerythroid progenitors of AM mice.Gas6 increases significantly the expression of EpoRand GATA-1 in cultured erythroid progenitors.In contrast,the Gas6 induction of EpoR andGATA-1 mRNA in vitro was completely blocked in AM erythroblasts.Notably,Gas6up-regulated the expression of EpoR and GATA-1 in the single mutant (Axl^-/- or Mer^-/-)erythroblasts.The results indicate that Axl and Mer may participate in erythropoiesis in redundant manner through regulating EpoR and GATA-1 expression.