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Transglutaminase activity in the hematopoietic tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
2008 (English)In: BMC immunology, ISSN 1471-2172, Vol. 9, no 58, 1-11 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Transglutaminases (TGases) form a group of enzymes that have many different substrates and among the most well known are fibrin for Factor XIIIa and the clotting protein in crustaceans. We also found that TGase is an abundant protein in the hematopoietic tissue (Hpt) cells of crayfish and hence we have studied the possible function of this enzyme in hematopoiesis. RESULTS: TGase is one of the most abundant proteins in the Hpt and its mRNA expression as well as enzyme activity is very high in the Hpt cells, lesser in the semi-granular hemocytes and very low in the granular cells. In cultured hematopoietic tissues, high activity was present in cells in the centre of the tissue, whereas cells migrating out of the tissue had very low TGase activity. RNAi experiments using dsRNA for TGase completely knocked down the transcript and as a result the cell morphology was changed and the cells started to spread intensely. If astakine, a cytokine directly involved in hematopoiesis, was added the cells started to spread and adopt a morphology similar to that observed after RNAi of TGase. Astakine had no effect on TGase expression, but after a prolonged incubation for one week with this invertebrate cytokine, TGase activity inside and outside the cells was completely lost. Thus it seems as if astakine addition to the Hpt cells and RNAi of TGase in the cell culture will lead to the same results, i.e. loss of TGase activity in the cells and they start to differentiate and spread. CONCLUSION: The results of this study suggest that TGase is important for keeping the Hpt cells in an undifferentiated stage inside the hematopoietic tissue and if expression of TGase mRNA is blocked the cells start to differentiate and spread. This shows a new function for transglutaminase in preventing hematopoietic stem cells from starting to differentiate and migrate into the hemolymph, whereas their proliferation is unaffected. Astakine is also important for the hematopoiesis, since it induces hemocyte synthesis in the Hpt but now we also show that it in some unknown way participates in the differentiation of the Hpt cells.

Place, publisher, year, edition, pages
2008. Vol. 9, no 58, 1-11 p.
National Category
Immunology
Research subject
Immunology
Identifiers
URN: urn:nbn:se:uu:diva-87490DOI: 10.1186/1471-2172-9-58ISI: 000260740500001PubMedID: 18840279OAI: oai:DiVA.org:uu-87490DiVA: diva2:132601
Available from: 2008-12-22 Created: 2008-12-19 Last updated: 2011-12-04
In thesis
1. Hematopoiesis in a Crustacean
Open this publication in new window or tab >>Hematopoiesis in a Crustacean
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hemocytes (blood cells) play an important role in the immune response in invertebrates, and thus the regulation of hemocyte homeostasis (hematopoiesis) is essential for the host survival against pathogens. Astakine 1, a homologue to vertebrate prokineticins, was first identified in the freshwater crayfish Pacifastacus leniusculus as a cytokine, and was found to be necessary for new hemocyte synthesis and release in vivo, and also to induce spreading and proliferation of Hematopoietic tissue cells (Hpt cells, precursor of hemocytes) in vitro. The work of this thesis is aimed to further our understanding of the molecular mechanisms involved in astakine 1 induced hematopoiesis.

Crayfish transglutaminase (Tgase) has been identified in the hemocytes, and is essential for the coagulation reaction. Interestingly this enzyme is exceedingly abundant in the Hpt cells, and the spreading of Hpt cells induced by astakine 1 was accompanied by sequential loss of TGase activity from the surface of these cells. This loss of TGase activity may be an important effect of astakine 1, resulting in recruiting new hemocytes into the circulatory system. Although astakine 1 contain a prokineticin domain, it lacks the conserved N-terminal AVIT motif present in its vertebrate homologues. This motif is important for vertebrate prokineticins to interact with their receptors, indicating a different receptor interaction for crayfish astakine 1. Astakine 1 was indeed found to interact with a completely different receptor, the β-subunit of ATP synthase, on a portion of Hpt cells, and subsequently block its extracellular ATP formation. Surface ATP synthase has been reported on numerous mammalian cells, but now for the first time in an invertebrate. The activity of ATP synthase on the Hpt cells may be important for the survival and proliferation of Hpt cells, but the underlying mechanisms remain further study. With the finding of a second type of astakine in crayfish, invertebrate astakines can be divided into two groups: astakine 1 and astakine 2. The properties of astakine 2 are different from those of astakine 1 both in structure and function. In primary cell culture of Hpt cells, only astakine 1 can promote proliferation as well as differentiation into semigranular cells, whereas astakine 2 may play a potential role in the maturation of granular cells. Moreover, a novel cysteine rich protein, Pacifastacus hematopoiesis factor (PHF), was found to be one target gene of astakine 1 in Hpt cells. Down regulation of PHF results in increased apoptosis in Hpt cells in vitro, and in vivo silencing PHF leads to a severe loss of hemocytes in the animal. Therefore astakine 1 acquires the anti-apoptosis ability by inducing its downstream gene PHF in the Hpt cells. With its ability to promote the survival, proliferation and differentiation of Hpt cells, astakine 1 is proven to be an important hematopoietic growth factor.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 46 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 723
Keyword
astakine, cytokine, hematopoiesis, prokineticin, transglutaminase, ATP synthase, innate immunity, apoptosis
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
urn:nbn:se:uu:diva-121000 (URN)978-91-554-7755-4 (ISBN)
Public defence
2010-05-05, Lindahlsalen, Evolutionary Biology Centre, Norbyvägen 18A, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-13 Created: 2010-03-17 Last updated: 2013-10-29

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Publisher's full textPubMedhttp://www.biomedcentral.com/1471-2172/9/58

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Söderhäll, KennethSöderhäll, Irene

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