Regulation of hematopoiesis in the freshwater crayfish, Pacifastacus leniusculus : role of transglutaminase

Abstract: The freshwater crayfish, Pacifastacus leniusculus, has been used as a model for studying hematopoiesis or blood cell production or hematopoiesis and immunity. The work of this thesis aims to investigate the impact of factors such as ROS signaling, Ast1, and the PVF/PVR signaling pathway in controlling stem cell behavior during hematopoiesis and specifically the role of the crosslinking enzyme transglutaminase (TGase) in regulation of hematopoiesis.The role of ROS in crayfish hematopoiesis was characterized by using the antioxidant named NAC to inhibit ROS production. Low ROS level resulted in a prolonged decrease in hemocyte numbers and a combined injection of LPS and NAC caused a slower rate of new hemocyte production. A low ROS level in cell cultures supplemented with crude Ast1 was found to inhibit cell spreading and a high extracellular TGase activity was detected on the surfaces of APC and HPT cells. We suggest that ROS serves as a prime signal to control proliferation and differentiation of progenitor cells by affecting extracellular TGase activity. We reported an inhibitory effect of Ast1 on TGase enzyme activity and on its crosslinking activity and consequently Ast1 affects the clot formation and thus coagulation by inhibiting the crosslinking activity of the TGase enzyme. Secretion of the clot protein (CP) and the production of CP filament network between spreading cells were observed in HPT cell cultures in vitro. In the presence of CP together with Ast1 in 3D-collagen-I cultures, HPT cells were found to be more elongated and they formed chains of cells throughout the surrounding matrix. In the HPT tissue, CP was located around the HPT cells or around the lobules of HPT, and thus, CP was demonstrated to be a part of ECM and to possibly function together with collagen in generating a suitable environment for HPT progenitor cells. The inhibition of PVF/PVR downstream signaling pathway by Sunitinib malate resulted in a dramatic change of cell morphology and induction of an increase cell surface area during cell culture. The addition of crude Ast1 into the cell cultures in vitro enhanced this effect. Consequently, cell migration was stimulated and a high extracellular TGase activity on HPT cell surface was found after this inhibition. In conclusion, the work in this thesis provides new insight in understanding the role of the extracellular matrix (ECM) and extracellular TGase activity in controlling stem cell activity.