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Secretion of 10-kDa and 12-kDa Thioredoxin Species from Blood Monocytes and Transformed Leukocytes
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0001-5082-6423
2000 (English)In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 2, no 4, 717-726 p.Article in journal (Refereed) Published
Abstract [en]

Thioredoxins (TRX) are ubiquitous, small redox-active proteins with multiple functions, including antioxidant, cytoprotective, and chemoattractant activities. In addition to a 12-kDa intracellular form, extracellular 10-kDa and 12-kDa TRX have been defined. The biological activities of the 10-kDa TRX were previously measured as eosinophil cytotoxicity enhancing activity or B-cell stimulatory activity. Cytotrophoblastic cell lines also release a 10-kDa TRX form. To study the biological role of 10-kDa TRX, we established two highly sensitive enzyme-linked immuno-spot assays (ELISPOT), which detect secreted truncated 10-kDa and full-length 12-kDa TRX at the single cell level. TRX secretion was investigated in several cell lines including the T-helper cell hybridoma MP6, the Jurkat T-cell leukemia, the U-937 myelomonocytic leukemia, and the 3B6, EBV-transformed, lymphoblastoid B-cell line. The highest number of secreting cells was found in 3B6 cultures, median = 34 (quartiles, 27–39) per well (105 cells). Peripheral blood monocytes isolated from healthy donors secreted significantly more TRX after stimulation with ionomycin, phorbol myristate acetate (PMA), fMLP, and lipopolysaccharide (LPS), compared to unstimulated cells. Oxidative stress induced by thioloxidant diamide also induced the secretion of both truncated and full-length TRX measured in ELISPOT (p = 0.047 and p = 0.031, respectively). The biological activity of the truncated and full-length forms was tested in a cell migration assay. Truncated TRX was devoid of protein disulfide reductase activity, but retained strong chemoattractant activity for human monocytes, in the same range as full-length TRX, as previously reported.

Place, publisher, year, edition, pages
2000. Vol. 2, no 4, 717-726 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25335DOI: 10.1089/ars.2000.2.4-717Local ID: 9777OAI: oai:DiVA.org:liu-25335DiVA: diva2:245663
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-09-22Bibliographically approved
In thesis
1. Thioredoxin system in normal and transformed human cells
Open this publication in new window or tab >>Thioredoxin system in normal and transformed human cells
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Thioredoxin (Trx) and thioredoxin reductase (TrxR), together with NADPH, constitute the Trx system, a major antioxidant entity that helps maintain a reducing environment within living cells. Trx designates a family of proteins that are related on the basis of structure and function. Human full-length Trx is a 12 kDa redox-active protein that contains the evolutionarily conserved active site sequence Cys-Gly Pro-Cys. Truncated Trx is a shorter, 10 kDa form of human Trx that shows complete homology to the N-terminal 80 or 84 amino acids of 12 kDa Trx. Truncated Trx displays no reducing activity, even though it contains an intact active site. Human TrxR is a homodimeric, FAD-containing, selenoprotein that reduces oxidized Trx back to the enzymatically active form by consuming NADPH. Expression of human Trx and TrxR is induced by a variety of stressors. The Trx system functions directly and indirectly in important biological features such as DNA synthesis, gene expression, co-cytokine activity, chemokine properties, scavenging reactive oxygen intermediates, apoptosis, and growth regulation.

Aims: The overall objective of this research was to examine the regulation and biological role of the Trx system in normal and transformed cells, and a possible connection with tumorigenesis. The specific aims were as follows: i) to study the presence and function of full-length and truncated Trx in normal and transformed human cells; ii) to assess the expression and function of TrxR; iii) to investigate the functional importance of truncated Trx; iv) to examine the regulatory mechanisms behind secretion of Trx, truncated Trx, and TrxR; v) to study full-length secreted Trx in human plasma.

Methods: Using Köhler and Milstein hybridoma technology, monoclonal antibodies against full-length and truncated Trx, as well as TrxR, were developed and applied in various immunological methods to detect the proteins in and secreted from normal and transformed human cells.

Results: Using selective monoclonal antibodies distinct intracellular localization of full-length and truncated Trx was shown. The latter was present in lower amounts, primarily associated with the plasma membrane; only small amounts of the 12 kDa were present on the plasma membrane. Moreover, we found that human TrxR was secreted from normal and transformed cells via a Golgi-dependent pathway. Using sensitive EUSPOT, TrxR release was quantified at the single-cell level and found to be inducible. TrxR was detected in plasma from healthy blood donors, and this protein was overexpressed in transformed cells, compared to their normal counterparts. Truncated 10 kDa Trx was present in and secreted from normal and transformed cells. Treatment of normal peripheral blood monocytes with LPS, IL-1, IFN-γ, PMA, ionomycin, and the thiol oxidant diarnide induced secretion of Trx. Trx was found within platelets, and liberation of this Trx was induced by diamide, implying a novel mechanism for release of Trx.

Conclusions: Monoclonal antibodies were generated against full-length and truncated Trx as well as TrxR and proved to be useful tools for investigating these proteins. Full-length and truncated Trx were found to have different cellular functions, suggesting that C-temrinal truncation regulates the activity of Trx. Secretion of TrxR and the presence of thls enzyme in plasma imply that the Trx system has both intracellular and extracellular functions. The presence of Trx in platelets indicates participation of the protein in coagulation and possibly also in the effects of platelets that maintain the reducing capacity of human plasma.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 86 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 642
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25707 (URN)10083 (Local ID)91-7219-743-9 (ISBN)10083 (Archive number)10083 (OAI)
Public defence
2000-10-13, Berzeliussalen, Hälsouniversitetet, Linköping, 09:30 (Swedish)
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Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-09-22Bibliographically approved

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