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Tissue Factor regulation, signaling and functions beyond coagulation with a focus on diabetes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Coagulation and inflammation science.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein best known for initiating the coagulation cascade upon binding of its ligand FVIIa. Apart from its physiological role in coagulation, TF and TF/FVIIa signaling has proved to be involved in diseases such as diabetes, cancer and cardiovascular diseases. Biological functions coupled to TF/FVIIa signaling include diet-induced obesity, apoptosis, angiogenesis and migration.

Aim: The aim of this thesis was to investigate the role of TF/FVIIa in cells of importance in diabetes, to further investigate the mechanism behind TF/FVIIa anti-apoptotic signaling in cancer cells and lastly to examine the regulation of TF expression in monocytes by micro RNAs (miRNA).

Results: In paper I we found that TF/FVIIa signaling augments cytokine-induced beta cell death and impairs glucose stimulated insulin secretion from human pancreatic islets. In paper II the relevance of TF/FVIIa in isolated human primary adipocytes was investigated. Adipocytes are a target cell for insulin and diabetics typically have increased lipolysis and impaired glucose uptake. No evidence was found for a role of TF/FVIIa in lipolysis or glucose uptake in adipocytes. However, adipocytes were found to express TF and FVII. The FVII produced was sufficient to initiate coagulation in the adipocytes. In paper III an anti-apoptotic TF/FVIIa induced signaling pathway in prostate and breast cancer cells was investigated in depth. Previous research has shown that TF/FVIIa signaling results in transactivation of insulin-like growth factor 1 receptor (IGF-1R) leading to subsequent protection from apoptosis induced by TNF-related apoptosis inducing ligand (TRAIL). The current results propose a mechanism where IGF-1R transactivation by TF/FVIIa is dependent on integrin β1 (ITGβ1) signaling. TF/FVIIa/ ITGβ1 signaling was found to result in phosphorylation of src and subsequent phosphorylation of caveolin 1 (Cav1). Once phosphorylated, the inhibitory effect of Cav1 on IGF-1R is cancelled, resulting in IGF-1R activation. In paper IV the role of miRNA regulation of TF expression in monocytic cells was investigated. The miRNA miR-223-3p was identified to be differentially expressed in U937 cells undergoing differentiation to a more monocyte-like phenotype and an anti-parallel correlation between TF and miR-223-3p expression in monocytes was proved. Hence, miR-223-3p regulates the inducible expression of TF in monocytes.

Conclusions: The work in this thesis furthers the knowledge of molecular mechanisms behind TF regulation and TF/FVIIa signaling and some functional consequences as well as their biological relevance in diabetes. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1626
Keywords [en]
Tissue factor, diabetes, cell signaling, coagulation, beta cells, adipocytes, apoptosis, lipolysis, micro RNA
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-399599ISBN: 978-91-513-0842-5 (print)OAI: oai:DiVA.org:uu-399599DiVA, id: diva2:1380668
Public defence
2020-02-21, Enghoffsalen, Akademiska sjukhuset, ing. 50, Uppsala, 13:15 (Swedish)
Opponent
Supervisors
Available from: 2020-01-30 Created: 2019-12-19 Last updated: 2020-03-25
List of papers
1. Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets
Open this publication in new window or tab >>Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets
2015 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, no 11, p. 2563-2572Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis Patients diagnosed with type 1 or type 2 diabetes have elevated levels of coagulation factor VIIa (FVIIa) and its receptor tissue factor (TF) in their bloodstream. This may affect the fate of the beta cells. We aimed to study the effects of TF/FVIIa signalling on cytokine-induced beta cell death and islet function in vitro. Methods Human pancreatic islets and MIN-6 beta cells were used to study TF mRNA and protein expression using real-time PCR, immunoblotting and flow cytometry. The effects of TF/FVIIa on cytokine-induced beta cell death were studied in MIN-6 cells and human pancreatic islets using cell-death ELISA and propidium iodide and cleaved caspase-3 staining. Effects of TF/FVIIa on the phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) were investigated by immunoblotting. Glucose-stimulated insulin secretion (GSIS) from human islets was measured with an insulin ELISA. Results A combination of the cytokines IL-1 beta, TNF-alpha and IFN-gamma induced TF expression in human pancreatic islets and in beta cells. TF/FVIIa did not affect basal beta cell death but, independently of downstream coagulation activity, augmented beta cell death in response to cytokines. The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death. Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets. Conclusions/interpretation These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines.

Keywords
Beta cells, Cytokines, Diabetes, FVIIa, Glucose-stimulated insulin secretion, JNK, Pancreatic islets, Tissue factor
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-265663 (URN)10.1007/s00125-015-3729-y (DOI)000361993000013 ()26271343 (PubMedID)
Funder
Göran Gustafsson Foundation for promotion of scientific research at Uppala University and Royal Institute of TechnologyStiftelsen Olle Engkvist ByggmästareMagnus Bergvall FoundationSwedish Society of MedicineSwedish Child Diabetes FoundationSwedish Research Council
Available from: 2015-11-05 Created: 2015-11-02 Last updated: 2019-12-19Bibliographically approved
2. Adipocytes express tissue factor and FVII and are procoagulant in a TF/FVIIa-dependent manner
Open this publication in new window or tab >>Adipocytes express tissue factor and FVII and are procoagulant in a TF/FVIIa-dependent manner
Show others...
2019 (English)In: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 124, no 3, p. 158-167Article in journal (Refereed) Published
Abstract [en]

Background: Tissue factor (TF) combined with its ligand FVII initiates blood coagulation and intracellular signaling. Obese and type 2 diabetic subjects have increased TF expression in their adipose tissue and an increased risk for thrombotic complications. Here we address the role of TF/FVII on adipocyte functions.

Materials and methods: Subcutaneous fat was obtained by means of needle aspiration from healthy volunteers, and adipocytes were isolated after collagenase digestion. 3T3-L1 fibroblasts kept in culture were differentiated into adipocytes by addition of IBMX, dexamethasone, rosiglitazone, and insulin to the media. Proteins and mRNA were analyzed by western blot and RT-PCR. Coagulation activity was determined by a colorimetric FX-assay. Lipolysis was measured as free glycerol using a colorimetric method. Glucose uptake was evaluated by scintillation counting of D-[U-C-14] glucose.

Results: In isolated human primary adipocytes we found expression of TF and FVII. TF expression was confirmed in 3T3-L1 adipocytes, and both cell types were found to be procoagulant in a TF/FVIIa-dependent manner. FXa was generated without FVIIa added to the coagulation assay, and active site-inhibited FVIIa blocked FXa formation, supporting our finding of FVII production by human primary adipocytes. There was no evidence for a role of TF in either lipolysis or glucose uptake in our experimental settings.

Conclusion: Human primary adipocytes express active TF and FVII, and the TF/FVIIa complex formed on the adipocyte surface can activate substrate FX. Whether the TF/FVIIa complex conveys signaling pathways leading to biological functions and has any biological activity in adipocytes beyond coagulation remains to be elucidated.

Place, publisher, year, edition, pages
TAYLOR & FRANCIS LTD, 2019
Keywords
Adipocytes, coagulation, FVII, lipolysis, tissue factor
National Category
Cardiac and Cardiovascular Systems Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-396115 (URN)10.1080/03009734.2019.1645248 (DOI)000481057900001 ()31407948 (PubMedID)
Funder
Swedish Research CouncilSwedish Heart Lung FoundationErik, Karin och Gösta Selanders Foundation
Available from: 2019-10-31 Created: 2019-10-31 Last updated: 2019-12-19Bibliographically approved
3. Activation of β1 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival
Open this publication in new window or tab >>Activation of β1 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-399437 (URN)
Available from: 2019-12-18 Created: 2019-12-18 Last updated: 2019-12-19
4. miR-223-3p regulates post-transcriptional tissue factor gene expression in human monocytic cells
Open this publication in new window or tab >>miR-223-3p regulates post-transcriptional tissue factor gene expression in human monocytic cells
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-399598 (URN)
Available from: 2019-12-18 Created: 2019-12-18 Last updated: 2019-12-19

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