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Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 34, 17593-17601 p.Article in journal (Refereed) Published
Abstract [en]

Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila. In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROS in both in vivo and in vitro experiments. An increase in ROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed.

Place, publisher, year, edition, pages
2016. Vol. 291, no 34, 17593-17601 p.
Keyword [en]
extracellular matrix, hematopoiesis, invertebrate, reactive oxygen species (ROS), transglutaminase
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
URN: urn:nbn:se:uu:diva-305568DOI: 10.1074/jbc.M116.741348ISI: 000383241300011PubMedID: 27339892OAI: oai:DiVA.org:uu-305568DiVA: diva2:1038601
Funder
Swedish Research Council, VR 20114797, VR 621-2012-2418
Available from: 2016-10-19 Created: 2016-10-19 Last updated: 2017-08-22
In thesis
1. Regulation of hematopoiesis in the freshwater crayfish, Pacifastacus leniusculus: role of transglutaminase
Open this publication in new window or tab >>Regulation of hematopoiesis in the freshwater crayfish, Pacifastacus leniusculus: role of transglutaminase
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1543
Keyword
Ast1, clotting protein, crayfish, hematopoiesis, PVF/PVR, ROS, transglutaminase
National Category
Cell Biology
Research subject
Biology with specialization in Molecular Biology; Biology with specialization in Comparative Physiology
Identifiers
urn:nbn:se:uu:diva-327921 (URN)978-91-513-0037-5 (ISBN)
Public defence
2017-10-06, Lindahlssalen, EBC, Norbyvägen 18A, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2017-09-12 Created: 2017-08-14 Last updated: 2017-09-12

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