Tissue Factor Regulation, Signaling and Functions Beyond Coagulation with a Focus on Diabetes

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1626

Tissue Factor regulation, signaling and functions beyond coagulation with a focus on diabetes

DESIRÉE EDÉN

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0842-5 UPPSALA urn:nbn:se:uu:diva-399599 2020 Dissertation presented at Uppsala University to be publicly examined in Enghoffsalen, Akademiska sjukhuset, ing. 50, Uppsala, Friday, 21 February 2020 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in Swedish. Faculty examiner: Docent, Universitetslektor Sofia Ramström (Örebro Universitet, Institutionen för medicinska vetenskaper).

Abstract Edén, D. 2020. Tissue Factor regulation, signaling and functions beyond coagulation with a focus on diabetes. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1626. 65 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0842-5.

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.

Keywords: Tissue factor, diabetes, cell signaling, coagulation, beta cells, adipocytes, apoptosis, lipolysis, micro RNA

Desirée Edén, Department of Medical Sciences, Akademiska sjukhuset, Uppsala University, SE-75185 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-513-0842-5 urn:nbn:se:uu:diva-399599 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-399599)

Till Leila, Jan och Robert

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Edén D, Siegbahn A, Mokhtari D. (2015) Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets. Dia- betologia, 58(11):2563–72 II Edén D, Panagiotou G, Mokhtari D, Eriksson JW, Åberg M, Sieg- bahn A. (2019) Adipocytes express tissue factor and FVII and are procoagulant in a TF/FVIIa-dependent manner. Upsala Journal of Medical Sciences, 124(3):158-167 III Åberg M, Edén D, Siegbahn A. Activation of β1 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival. Manuscript IV Alfredsson J, Edén D, Christersson C, Åberg M, Siegbahn A. miR- 223-3p regulates post-transcriptional tissue factor gene expression in human monocytic cells. Manuscript

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 11 Tissue factor and FVIIa ...... 13 Background ...... 13 TF/FVIIa initiation of the coagulation process ...... 13 Blood borne tissue factor ...... 14 TF expression ...... 15 miRNA ...... 16 TF/FVIIa intracellular signaling ...... 18 PAR signaling ...... 19 RTK signaling ...... 20 Integrin signaling ...... 21 Diabetes ...... 23 Background ...... 23 The immune system and cytokines ...... 24 Classification of diabetes ...... 24 T1D and T2D ...... 25 Adipocyte function and malfunction in T2D ...... 26 TF and FVIIa in diabetes ...... 28 Methods ...... 29 Cell culture and isolated cells ...... 29 Protein expression and cell signaling ...... 30 Western blot ...... 30 Flow cytometry ...... 30 Protein proximity ligation (PLA) ...... 30 Gene expression ...... 31 siRNA gene silencing ...... 31 qRT-PCR ...... 31 RT-PCR ...... 31 Functional studies ...... 32 Apoptosis ...... 32 Glucose stimulated insulin secretion (GSIS) ...... 32 Lipolysis ...... 32 Glucose uptake ...... 33 TF coagulation activity ...... 33

Aims ...... 34 Results and Discussion ...... 35 Paper I ...... 35 Background ...... 35 Results and discussion ...... 35 Paper II ...... 37 Background ...... 37 Results and discussion ...... 37 Paper III ...... 39 Background ...... 39 Results and discussion ...... 39 Paper IV ...... 41 Background ...... 41 Results and discussion ...... 41 General discussion and future perspectives ...... 43 TF/FVIIa in diabetes ...... 44 The potency of TF/FVIIa coagulation initiation ...... 46 The complexity of TF/FVIIa intracellular signaling transduction ...... 46 miRNA ...... 48 Conclusions ...... 50 Populärvetenskaplig sammanfattning på svenska ...... 51 Acknowledgments ...... 55 References ...... 57

Abbreviations

APC Antigen presenting cells asTF Alternatively spliced tissue factor AT-III Antithrombin III Cav1 Caveolin 1 EGFR Epidermal growth factor receptor FFA Free fatty acid FVIIa Active coagulation factor VII FVIIai Active site-inhibited coagulation factor VII FVII Coagulation factor VII GADA Glutamic acid decarboxylase antibodies GPCR G protein-coupled receptors GSIS Glucose stimulated insulin secretion IFN-γ Interferon γ IGF-1R Insulin-like growth factor 1 receptor IL-1 Interleukin 1 IR Insulin receptor ITGβ1 β1 integrin JNK C-Jun N-terminal kinase kDa Kilo Dalton LADA Latent autoimmune diabetes in adults LPS Lipopolysaccharide MAPK Mitogen-activated protein kinases miRNA Micro ribonucleic acid mRNA Messenger ribonucleic acid MV Micro vesicles NF-κB Nuclear factor-kappa B PAR Protease-activated receptor PDGFRβ Platelet-derived growth factor receptor β PI3K Phosphatidylinositol-3 kinase PLA Proximity ligation assay qRT-PCR Real-time quantitative polymerase chain reaction RTK Receptor tyrosine kinase RT-PCR Reverse transcriptase polymerase chain reaction SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TF Tissue factor

TFPI Tissue factor pathway inhibitor TNF-α Tumor necrosis factor α TRAIL TNF-related apoptosis inducing ligand T1D Type 1 diabetes T2D Type 2 diabetes

Introduction

The process of blood coagulation with thrombus formation is of essence in order to prevent major bleeding and blood loss in case of an injury. At the same time, it is of equal importance that the blood does not coagulate and form thrombus inside intact vessels. In order to prevent both major blood loss and potential fatal thrombus formation, the coagulation system is a complex and well-orchestrated system with many components. In addition to thrombus formation, the components of the coagulation system are involved in other biological functions unrelated to coagulation.

Tissue factor (TF) is best known as the main initiator of blood coagulation but has also been proved to function as a signaling receptor with effects unrelated to the process of clot formation. There is data supporting a role for aberrant TF expression in diseases associated with thrombosis such as cardio vascular disorders (CVD), diabetes and cancer [1-3]. There is also accumu- lating evidence for non-coagulant TF signaling being involved in apoptosis, inflammation and migration [4-6].

Micro RNAs (miRNAs) are small non-coding RNAs. miRNAs play a role in post-transcriptional regulation of protein expression by preventing translation of messenger RNA (mRNA). Several miRNAs have been suggested to regulate the expression of TF [7]. In paper IV, the role of the miRNA miR- 223-3p in regulation of TF expression is investigated in cell models of monocytes as well as in purified human monocytes.

Cancer is associated with an increased risk for thrombosis and several types of cancer cells express TF [2]. Apart from an increased risk for thrombotic events, there is also evidence for TF signaling playing a role in migration and survival of cancer cells [8-10]. In paper III, the detailed mechanism of an anti-apoptotic pathway is proposed in cancer cell models of prostate and breast cancer.

Insulin is produced and released by the beta cells, which are located in Langerhans islet in the endocrine part of pancreas. The hormone is responsible for cellular glucose up-take and released to the blood as a response to increased blood-sugar levels, thereby maintaining blood-sugar homeostasis.

11 [11]. Diabetes is an increasing problem worldwide and characterized by increased blood glucose levels due to cells failing to take up glucose. This can either depend on insufficient insulin production, peripheral insulin resistance or both [12].

Diabetic patients have elevated plasma levels of TF antigen and its ligand coagulation factor VIIa (FVIIa) and also have increased coagulation activity. TF expression is e.g. up regulated in the adipose tissue of type 2 diabetic (T2D) patients and obese subjects [13, 14]. This unspecific connection between TF/FVIIa and diabetes is well established, but the function of TF in cells and tissues that play important roles in diabetes has not been sufficient- ly investigated. Paper I and II in this thesis investigates the contribution of TF/FVIIa signaling in cells with known impaired function in diabetes, i.e. beta cells and adipocytes.

12 Tissue factor and FVIIa

Background TF is a 47 kDa transmembrane glycoprotein of the type II cytokine receptor family and the cell surface receptor for FVII. The binding of FVII leads to its activation into FVIIa and formation of the binary TF/FVIIa complex and the subsequent initiation of the coagulation process. TF consists of 263 amino acids; residue 1-219 constitutes the extracellular domain, residue 220-242 the transmembrane part and finally residue 243-263 constitutes the cytoplasmic domain [15-17]. Unlike many other cytokine receptors, the cytoplasmic domain of TF lacks tyrosine kinase recruitment motifs. Instead, it can be phosphorylated on either of its three serines and become involved in e.g. regulation of integrin function and cell migration [18, 19]. TF extracellular part has two disulphide bonds and is glycosylated in three residues [15- 17]. When TF is oxidized, the disulphide bonds are intact and TF is in its coagulation active, or decrypted, form. When TF is reduced the disulphide bond is broken and TF is in its coagulation inactive, or cryptic, form. Thus, it has been suggested that the disulfide bond Cys186-Cys209 in TF is an allosteric bond and thereby controls TF procoagulant activity.

TF/FVIIa initiation of the coagulation process TF is normally expressed on cells that are not in contact with flowing blood and the distribution functions as a hemostatic envelope. Examples of cells with a constitutive TF expression are sub-endothelial cells (e.g. smooth muscle cells) and cells surrounding blood vessels and blood rich organs such as the brain, lung, kidney and placenta (e.g. fibroblasts) [4, 20]. Cells inside the vessels, in direct contact with the blood, such as endothelial cells and monocytes do not normally express TF. However, TF expression can be induced in these cells by various stimuli such as inflammatory mediators, direct contact with leucocytes or small circulating extracellular vesicles or by lipopolysaccharide (LPS) from gram-negative bacteria [21, 22]. LPS are frequently used in experimental settings to induce TF expression in vitro.

In healthy individuals, TF is exposed to the blood and its ligand FVII upon vascular injury (Figure 1). FVII is an inactive serine protease precursor that

13 becomes the active protease FVIIa by cleavage of a peptide bond when binding to TF. TF/FVIIa in turn activates factor IX and factor X to generate a small amount of thrombin and this is known as the initiation phase, the first of three phases in the coagulation cascade. The initiation phase is followed by the amplification phase where platelets and cofactors are being activated to provide a negative surface of phospholipids and the tenase and prothrombinase complexes. The activation of platelets and cofactors furthers the coagulation process to the third and final phase, the propagation phase. In the propagation phase, the burst of thrombin generated by the prothrombinase complexes leads to cleavage of a sufficient amount of fibrinogen to fibrin to form a stable clot [23]. TF is most active during the initiation phase and its activity is shut down by tissue factor pathway inhibitor (TFPI) [24].

Figure 1. The cell bound model of coagulation. Blood coagulation begins by binding of FVIIa to TF. Platelets are recruited and adhere to the site of injury and the TF/FVIIa complex further activates the coagulation factors IX to IXa and X to Xa and a trace amount of thrombin is generated. The small amount of thrombin leads to platelet activation and aggregation and activates factor V, factor VIII and factor XI on the platelet surface. Next, activated factor IX binds to factor VIIIa to provide FXa. Factor Xa cannot be transported from the TF-expressing cell due to rapid inhibition by the protease inhibitors tissue factor pathway inhibitor and antithrombin. When factor Xa associates with platelet factor Va it is protected from the protease inhibitors. Upon this association, a burst of thrombin is generated, which is sufficient for the cleavage of fibrinogen and formation of the fibrin meshwork. Finally, a stable thrombus is formed.

Blood borne tissue factor In its full-length form TF is a cell-bound protein but it can still be found in the circulation in different settings. In an alternatively spliced isoform of TF (asTF), exon 5 is skipped and this leads to a frame-shift mutation. The result of this frame-shift is an isoform of TF that lacks the transmembrane region,

14 has a truncated extracellular domain, a unique carboxyl terminus and is soluble instead of anchored to the cell membrane [25]. asTF is not considered pro-coagulant but instead ligates endothelial integrin and regulates angiogenesis and monocyte recruitment [26, 27].

Extracellular vesicles (EVs) consist of a heterogeneous population of vesicles ranging from 0.03 to 1 µm in size that are released from cells by membrane budding (micro vesicles, MV) or exocytosis (exosomes) [28]. EVs are shed from almost all cell types [29] and are in general not considered procoagulant. The pro-coagulant activity is however increased if the EVs are shedded from a TF expressing cell and TF is present on the surface of the circulating EV [30-32]. In healthy individuals, the levels of TF positive EVs are undetectable or low, but they increase in a variety of diseases and has the potential to serve as biomarkers for thrombosis [30]. Additionally, there is also a shedded form of TF generated by proteolytic cleavage. This form does not take part in the coagulation since it is no longer bound to a phospholipid surface.

TF expression Our genetic information is stored in the DNA in the nucleus of every cell. The DNA is a molecule that consists of two strands that coil around each other to form a double helix structure. The backbone of the double helix is built up by sugar and phosphate whereas the nucleobases form base pairs and keep the two chains together. A gene is a part of DNA and can vary in size from a few hundred to up to a million DNA bases. The DNA and hence the genetic information, is the same in all cells in the human body, what makes the cells different from one another is what information, which genes, are being expressed and which are not.

Some genes are coding for proteins. In order for a protein to be expressed the corresponding gene must first be expressed in a process called transcription. Put simply; a gene coding for proteins is transcribed into mRNA with the help of the protein polymerase II. mRNA are a single-stranded molecule with base pairs corresponding to the gene that are being transcribed. mRNA carry the information from the gene and are transported from the nucleus of the cell to the cytoplasm. In the cytoplasm, ribosomes attach to the mRNA and translate the information to form a protein.

TF is a protein and its gene is transcribed into an mRNA, as described above. The TF gene, F3, is located on the short arm of chromosome 1. It is composed of 6 exons and 5 introns that covers almost 12.6 kbp of genomic sequence. Exon 1-5 corresponds to the extracellular part of TF and exon 6 to

15 the transmembrane and intracellular part [33, 34]. The expression of TF is regulated by two alternative transcription factor binding sites within the promoter region. The proximal promoter region regulates constitutive TF expression whereas the distal promoter region regulates inducible TF expression [33]. The F3 gene can be transcribed from either of these promoter regions and the result is a 2.2 kbp mature mRNA transcript [35].

Even though the TF gene is transcribed and an mRNA is formed, this does not necessarily mean that the TF protein itself is expressed. One of the mechanisms that regulate mRNA translation is micro RNAs (miRNAs). miRNA miRNAs are small, non-coding RNA molecules meaning that they, in contrast to mRNA, are not translated into proteins. Their best-studied function is instead to regulate the expression of proteins post transcription by silencing mRNAs and prevent them from being translated into proteins (Figure 2). miRNAs are present in cells, tissues and as stable entities associated with protein-complexes and extracellular vesicles in body fluids such as plasma. Genes for miRNA are transcribed like other genes, with the help of polymerase II, but in contrast to protein coding genes, the result is a primary miRNA that forms a typical hairpin loop. The primary miRNA is processed in multiple steps; first it is cleaved by DCGR8 and the RNAse III Dorsha to form a smaller precursor miRNA. The precursor miRNA is carried to the cytoplasm by the transporter Exportin 5. In the cytoplasm, the precursor miRNA is further processed by the RNAse III Dicer. One strand of the miRNA is shed off and the other strand forms a complex with an Argonaute protein and forms the RISC (RNA Induced Silencing Complex) [7, 36, 37]. The strand of the miRNA is complementary to that of a target mRNA, enabling base pairing. There are multiple ways that RISC can inactivate the mRNA. One way is to simply cut the mRNA that is then destroyed; another way is to inhibit translation by hindering binding of the ribosome subunit. The result is prevention of translation and the gene is silenced [36].

Figure 2. miRNA mediated gene silencing. miRNA is transcribed from DNA with the help of polymerase II. The transcribed primary miRNA forms a typical hairpin loop and is further cleaved to form the precursor miRNA. The precursor miRNA is transported to the cytoplasm where it is processed into smaller entities and one single strand of miRNA associates with proteins to form the RNA Induced Silencing Complex (RISC). The single strand miRNA is complementary to a target site of mRNA and RISC binds to the mRNA with base pairing. When bound to the target site of mRNA, RISC inactivates the mRNA, either by cutting the mRNA that then gets degraded or by hindering the ribosome subunit from binding. Thereby, translation of mRNA is prevented and the gene is silenced.

Biological relevance of miRNAs and its possible applications in healthcare miRNAs play an important role in regulation of protein expression in healthy individuals as well as in diseases. Their biological functions include a variety of aspects such as developmental timing, cell death and proliferation, hematopoiesis and tumorigenesis [38]. miRNAs have been found to be differentially expressed and detectable in circulation in many diseases such as Alzheimer’s disease [39-41], Parkinson’s disease [36], gastric cancer [42], breast cancer [43], T2D [44] and coronary artery disease [45] and could possibly serve as clinical biomarkers for these diseases. In addition to being possible biomarkers, miRNAs are also being explored both as targets for treatment and as therapeutics for diseases. For instance, studies are ongoing for the use of miRNAs as therapeutics and targets in different cancers [46, 47] and as therapeutics in Parkinson’s disease [36].

Regulation of TF expression by miRNA Several miRNAs have been shown to directly regulate TF protein expression and thereby its functions in different cell types and tissues. This can impact physiological functions as well as pathophysiological conditions [7]. For

17 instance, miR-19b and miR-20a reduce the expression of TF in vitro and is at the same time found to be lower in monocytes from patients with systemic lupus erythematosus and antiphospholipid syndrome. This indicates a role for miRNA regulation of TF in these diseases [48]. In a similar manner, miR-19 reduces the expression of TF in breast cancer cells and the data indicate that miRNA regulation of TF may affect tumor-associated functions [49]. A more direct impact of TF function by miRNA has been shown in colon cancer cells where miR-19a suppressed TF expression and its mediated migration and invasion [50]. One study reports that miR-126 exhibits antithrombotic properties by targeting TF expression and thereby impacting the hemostatic balance of the vasculature in diabetes mellitus. miR-126 could be a possible prognostic biomarker for development and complications in diabetes mellitus [51].

TF/FVIIa intracellular signaling Independent from its role in coagulation, TF also functions as a signaling receptor [52]. This was first discovered in 1995 when Rottingen et. al. reported that FVIIa addition to numerous TF-expressing cell types induced Ca2+ oscillation [53]. TF signaling can be mediated by the binary TF/FVIIa complex and by the ternary TF/FVIIa/FXa complex. The signaling can be dependent or independent of the protease activated receptors (PAR) PAR2 and PAR1, the TF cytoplasmic domain, by activation of integrins and through transactivation or proteolytic cleavage of members of the receptor tyrosine kinases (RTKs) (Figure 3).

Figure 3. Initiation of TF signaling on the cellular surface. Cell surface bound full length TF interacts with a number of different proteins in order to initiate signaling and achieve specificity. A) PAR1 is cleaved by the TF/FVIIa/FXa complex whereas PAR2 is cleaved by both TF/FVIIa and TF/FVIIa/FXa. TF cytoplasmic domain is Ser253 phosphorylated by PKCα and Ser258 phosphorylated by P38α. B) Associa- tion of TF with β1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. C) EphB2 and EphA2 are proteolytic targets of TF/FVIIa. D) TF/FVIIa transactivates the PDGFRβ in a PAR2 and Src-family. Image from Åberg et al, Semin Thromb Hemost 2015; 41(07):691-699 © Georg Thieme Verlag KG

Signaling pathways known to be activated by TF/FVIIa includes mitogen- activated protein kinases (MAPK); p44/42, p38 and C-Jun N-terminal kinase (JNK) that are mainly responsible for control of cell cycle and PI3K/AKT that are mainly responsible for cell survival. Events up-stream of both p44/42 and PI3K/AKT involve Src-family kinases [54-57]. Biological functions coupled to TF/FVIIa signaling include cell survival and migration as well as inflammation and angiogenesis in cancer cells [6, 8-10, 58].

PAR signaling PARs is a family of G protein-coupled receptors (GPCRs) that was first discovered in 1991 when the thrombin receptor was cloned [59]. There are four known PARs in humans that are widely expressed in many cell types. The PARs are not activated by endogenous extracellular agonists like most

19 GPCRs but are instead specifically cleaved by proteolytic enzymes, such as serine proteases, near the N-terminus. The proteolytical cleavage unmasks a tethered ligand that then folds back and binds to specific regions to activate the receptor [59]. PAR1 is the main thrombin receptor, but PAR3 and PAR4 can likewise be cleaved and activated by thrombin. In addition to thrombin, PAR1 can be cleaved and activated by the TF/FVIIa/FXa complex. PAR1 cleavage by TF/FVIIa/FXa can lead to Ca2+ influx and p44/42 MAPK activation in a process independent of the TF cytoplasmic domain [60, 61].

In the context of TF/FVIIa signaling, PAR2 is perhaps the most interesting of the PARs. Unlike the other PARs, PAR2 is thrombin-insensitive and can be activated by direct cleavage by the binary TF/FVIIa complex as well as by the ternary TF/FVIIa/FXa complex. Moreover, PAR2 signaling can be dependent on the cytoplasmic domain of TF [62-64]. For cleavage of PAR1/PAR2 by the ternary TF/FVIIa/FXa complex, the same low picomolar range concentrations of FVII is required as for activation of coagulation. For PAR2 to be activated by the binary TF/FVIIa complex, much higher concentrations are needed [65, 66]. When PAR2 is activated by the binary TF/FVIIa complex, the signaling can be dependent on phosphorylation of the TF cytoplasmic domain [67]. TF/FVIIa activation of PAR2 gives a pro- inflammatory and pro-angiogenic response, resulting in secretion of cytokines and angiogenic factors of importance for inflammatory, respiratory, gastrointestinal, metabolic, cardiovascular, and neurological diseases, as well as cancers [5]. Moreover, TF-PAR2 signaling in hematopoietic and myeloid cells has been suggested to drive insulin resistance and inflammation in adipose tissue [18].

RTK signaling RTKs are a large class of cell surface receptors, with approximately 20 different subclasses [68]. The first RTKs to be discovered were the epidermal growth factor receptor (EFGR) and the neurotrophic growth factor receptors (NGFR) in the 1960s [69]. All RTKs consist of a transmembrane region, an N-terminal extracellular domain and a C-terminal cytoplasmic domain. RTK ligands include growth factors, hormones and cytokines. When a ligand binds to the extracellular domain of the receptor, dimerization of subunits are induced and subsequent tyrosine phosphorylation and auto phosphorylation of the cytoplasmic domain follows. The initiated downstream signaling transduction pathways include e.g. ras/MAPK and PI3K/AKT. RTK signaling play a key regulatory role in normal cellular processes but also has a critical role in the development of many diseases, including cancer [70, 71].

In addition to ligand-activation, RTKs can also be transactivated. Transacti- vation refers to the process of where activation of one receptor leads to the

20 activation of another receptor. One typical example is GPCR ligand activation of RTKs. Compared to ligand activation, transactivation typically gives a weaker response and the outcome of downstream signaling might be modi- fied by GPCR mediators [72].

EphB2 and EphA2 of the Eph RTK family have been proved to be proteolytical targets of TF/FVIIa. TF/FVIIa can cleave these receptors in their ecto- domains and thereby affect Eph-mediated cell segregation [73]. Apart from directly interacting with RTKs on the cell surface as described for the Eph receptors, TF also initiates RTK transactivation. To date, three RTKs are known to be transactivated by TF/FVIIa: EGF receptor (EGFR) [74], PDGF receptor β (PDGFRβ) [75], and insulin-like growth factor 1 receptor (IGF- 1R) [76]. The EGFR transactivation involves stimulation of metalloprotein- ases, which subsequently release heparin bound EGF to activate the receptor, a mechanism named the triple membrane-spanning model (TMSP). The transactivation may also be mediated by intracellular protein kinases. We identified for the first time the TF/FVIIa-induced transactivation of the PDGFRβ with a distinct pattern of phosphorylation at four tyrosines in the PDGFRβ cytodomain. This mechanism was shown to be PAR-2 dependent [75]. In contrast, transactivation of the IGF-1R is independent of PARs [76]. The tyrosine phosphorylation responses by TF/FVIIa are lesser in magnitude as compared to the receptors native ligands, which indicates a partial activation of the receptors and specificity in the activation.

While the biological consequences of EGFR transactivation are less clear, both PDGFRβ and IGF-1R transactivation by TF/FVIIa have major impacts on the target cells. Smooth muscle cells, fibroblasts, monocytes and endothelial cells are all sensitized by TF/FVIIa to migrate towards a 100-fold lower concentration gradient of PDGF-BB than cells without TF/FVIIa complex formation, owing to activation of Src-family kinases and transactivation of PDGFRβ [74, 75]. The IGF-1R, on the other hand, regulates cell survival. TF/FVIIa has been shown to have anti-apoptotic effects, mediated through transactivation of IGF-1R. This was demonstrated by the fact that both specific IGF-1R inhibitors and siRNA-mediated knockdown of the IGF-1R abolished the TF/FVIIa-dependent protection against apoptosis in breast and prostate cancer. The IGF-1R is also translocated to the nucleus after the transactivation where it binds to DNA and activates gene transcription [76].

Integrin signaling Integrins are transmembrane cell surface receptors that exert important biological functions such as adhesion of cells to extra cellular matrix (ECM), cell-to-cell adhesion and cell migration. Integrins are heterodimers that consist of two subunits, one α subunit and one β subunit. There are several dif-

21 ferent α- and β subunits and the combinations of them make up around 24 unique integrins [77, 78]. In coagulation, integrins on the surface of platelets are responsible for the attachment of fibrin within a developing thrombus [79].

In addition to the biological functions already mentioned, integrins play an important role in cell signaling by modulating the cell signaling pathways of e.g. IGF-1R [80]. TF/FVIIa can signal through interaction with other cell surface receptors, including PARs and RTKs, but also through interaction with integrins [81, 82]. As mentioned above, in TF/FVIIa dependent PARs and Eph receptor signaling there is a proteolytic cleaving to activate the receptors. In contrast, β1 integrins (ITGβ1) are not activated by proteolytical cleavage by TF/FVIIa. Instead, the mechanism for ITGβ1 activation by TF/FVIIa seems to relay on a more direct physical interaction between the complexes [81, 82]. An integrin-binding motif in the FVIIa protease domain has been described and is required for the association of TF/FVIIa with ITGβ1. In the light of this, TF can be described as a co-factor/scaffold to FVIIa which in turn interact with ITGβ1 and induce the conformational changes leading to ITGβ1 activation [82]. The resulting TF/FVIIa/ITGβ1 signaling has been coupled to tumor growth and pro-angiogenesis [81, 82].

22 Diabetes

Background Put shortly; diabetes is an inability of cells to take up glucose from the blood. This results in high blood sugar levels with symptoms such as increased thirst and hunger and frequent urination. Complications of diabetes include cardiovascular disease, stroke, renal failure, nerve damage, blindness and even death [12, 83]. Diabetes was first described in an Egyptian manuscript from 1500 BCE. The first descriptions of diabetes most likely describe what we today know as type 1 diabetes (T1D), with physicians of the time describing patients with large amount of urine with a sweet smell and taste. Indian physicians Sushruta and Charaka, around 400-500 CE, are the first known to have described T1D and T2D as two different conditions with T1D associated with youth and T2D associated with obesity [84].

Diabetes was linked to the pancreas in 1889, and more specifically to the islets of Langerhans in 1901 [85, 86]. The connection between diabetes and islets of Langerhans lead to researchers trying to isolate the secretion from the islets of Langerhans in search for a treatment of diabetes. In 1921, Fred- erick Banting managed to isolate what he then called “isletin” (today known as insulin) and proved its role in diabetes [87]. Continous research by Bant- ing together with Charles Best and in collaboration with J.B. Collip lead to the successful treatment of diabetes with insulin [88].

Insulin is a peptide hormone, produced by the beta cells located in the endocrine part of pancreas, more specifically the pancreatic islets of Langerhans. Insulin plays a central role in diabetes mellitus. It is normally released from the beta cells in response to high blood sugar and function as the main regu- lator of glucose uptake in muscle, liver and fat cells. An insufficient production of insulin or insulin resistance in peripheral cells therefore leads to glucose not being taken up by cells. This results in high levels of glucose in the blood, known as hyperglycaemia [11].

23 The immune system and cytokines The immune system plays important roles in the underlying mechanisms of both T1D and T2D [89-92]. In addition, and with relevance to this work, TF/FVIIa signaling has been coupled to inflammation, which is an important function of the immune system [5, 18, 89, 93]. The immune system is devel- oped to protect against microorganisms and is, like the coagulation system, a complex and well-orchestrated system that includes several cell types and signaling molecules. It is divided in the innate immune system and the adaptive immune system.

The innate immune system is the first line in host defense. If invaded by a foreign microorganism, leukocytes will recognize and respond to common pathogen proteins [94]. Leukocytes, e.g. macrophages, will release cytokines like IL-1 and TNF-α. This will result in local inflammation but also initiate the systemic acute response syndrome as well as the adaptive immunity through antigen presenting cells (APC) [94].

The adaptive immune system is, in contrast to innate immunity, specific and characterized by antigen specificity and memory capacity [94]. T- lymphocytes play a central role in adaptive immunity and are a major source of cytokines. T-lymphocytes are divided based on cell surface molecules into CD4+ and CD8+ T-lymphocytes. CD4+ are also known as helper T-cells and further divided into Th-subsets [95]. T-lymphocyte specificity is based on what antigen is presented by APCs and secondary signals, i.e. innate cytokines, determine what Th-subset they will develop into. There are several different Th subsets that produce different cytokines and have different ef- fector responses [94]. Th1 and Th2 cytokines often has counteracting effects and a balance between the two is most likely beneficial. Th1 produce pro- inflammatory cytokines like TNF-α and IFN-γ. Th2 produce anti- inflammatory cytokines like IL-10 and IgE-promoting cytokines like IL-4, IL-5 and IL-13. A too strong Th1 response can result in autoimmunity whereas a too strong Th2 response is associated with allergies [95]. Destruc- tive insulitis, as seen in T1D, is associated with a Th1 cytokine profile and T1D is regarded as an autoimmune disease [90, 91].

Classification of diabetes As mentioned above, Sushruta and Charaka described T1D as being associated with youth and T2D as being associated with obesity already 400-500 CE and broadly, diabetes is still classified into these two main types. In more recent years however, the landscape of diabetic subgroups has been re-drawn with the help of refined methods such as genetics and data-driven cluster

24 analysis [96-98]. One subgroup is the latent autoimmune diabetes in adults (LADA) that affects less than 10% of the diabetes population. LADA is defined by the presence of glutamic acid decarboxylase antibodies (GADA) and phenotypically presents as T2D at diagnosis but over time becomes more similar to T1D [99]. There are also rare, monogenic forms of diabetes described like maturity-onset diabetes of the young and neonatal diabetes [97, 98]. Groop et. al. [96] used the above mentioned cluster analysis to further divide patients with adult-onset diabetes into subgroups. Clusters were based on six variables; GADA, age at diagnosis, BMI, HbA1c, beta-cell function and insulin resistance. This method identified five different clusters, or subgroups, and could hopefully be used clinically in the future to identify patients at higher risk of diabetes complications, provide information about underlying mechanism and thereby guide therapy choice. Although there is a grey-zone between T1D and T2D and further subgroups exists, the simplified definition into T1D and T2D will further on be used in this thesis. Thus, T1D is defined as diabetes with presence of autoantibodies against pancreatic islet beta cell antigens and younger age at diagnosis. T2D is defined as diabetes with absence of autoantibodies against pancreatic beta cell antigens. Using this definition, T1D represents approximately 5-10% of all cases of diabetes and T2D represents the remaining 90-95%.

T1D and T2D T1D is described as an autoimmune disease and is today most often treated with insulin replacement [100]. There is a genetic component of T1D with monozygotic twins showing about 50% concordance rate in developing of the disease and the risk for a first degree relative to develop T1D is approximately 5% [101]. Although there is a genetic factor it clearly does not on its own explain the disease development, which is why the involvement of environmental factors has been investigated. So far, viral infections, toxins and early infant diet has been identified as possible contributors for the incur- rence of T1D [102, 103].

T1D occurs when the insulin producing beta cells located within the pancreatic islets of Langerhans becomes dysfunctional, gets destructed and die. This is most likely initiated by infiltrating immune cells causing a local inflammation. The first cells to invade the islets are macrophages, followed by CD4+ and CD8+ T-lymphocytes [93]. The beta-cell destruction is believed to be a result of direct cell-cell contact with the infiltrating cells and exposure to pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-γ released from these infiltrating immune cells [100, 104].

T2D is, in contrast to T1D, described as a lifestyle disease that correlates with a westernized diet and inactivity. Treatment of T2D in early stages of-

25 ten includes lifestyle interventions like diet and exercise. Bariatric surgery for weight loss is also discussed as a treatment for T2D [105]. First line med- ication often aims at preventing insulin resistance and inflammation and to protect beta cell function. Not until advanced stage of the disease is it common to get insulin replacement [106]. The pathogenesis is more variable in T2D than in T1D and, in addition to beta cell death or failure, also include peripheral insulin resistance [107, 108]. Insulin resistance can have many causes but the far most common one is obesity [109]. T2D is, like insulin resistance, associated with obesity [84]. A simplified model is that obesity leads to insulin resistance, which in turn cause T2D. Over the last decades, obesity has become a growing problem, not only in westernized countries but globally [110]. As of 2016, the world health organization estimated that worldwide, 39% of adults were overweight and 13% were obese. Over- weight is defined as a body mass index (BMI) of 25 or greater and obesity is defined as a BMI of 30 or greater. Since 1975, worldwide estimated obesity has nearly tripled and the prevalence of overweight and obese children and adolescents is on a steady increase. Obesity is associated with increased morbidity, mortality and comorbidities such as hypertension, dyslipidemia and the before mentioned T2D, all of which are risk factors for a plethora of cardiovascular and thrombotic complications [18, 111].

Adipocyte function and malfunction in T2D Insulin resistance is defined as inhibition of insulin stimulation of metabolic pathways including glucose transport, glycogen synthesis and anti-lipolysis [112]. As mention above, insulin resistance is linked to T2D and commonly caused by obesity. Exactly how obesity cause insulin resistance is not under- stood but most likely involve excessive nutrient intake and expanded adipose tissue [109]. Adipose tissue was long thought of as an inactive storage site for excessive calories in form of fat. Today, adipose tissue is described as a highly active metabolic tissue and endocrine organ [109]. The expansion of adipose tissue that occurs with weight gain is a result of adipocytes both increasing their size (hypertrophy) and increasing in number (hyperplasia) [113]. Hypertrophy of adipocytes is associated with insulin resistance and an adipocyte volume threshold over which T2D risk increases rapidly have been suggested [114].

Individuals with larger adipocytes typically have elevated pro-inflammatory factors and increased lipolysis in contrast to individuals with smaller adipocytes [115, 116]. This dysfunction of adipocytes, with hypertrophic adipocytes as the initiator, may induce systemic inflammation. An activation of the immune system is typically seen in obesity, with both higher levels of circulating pro-inflammatory cytokines and with infiltration of immune cells

26 like macrophages in e.g. adipose tissue and pancreas [89]. Macrophages act as sensors for alterations in the local immune environment of obese adipose tissue. During development of obesity, immune cell composition of adipose tissue changes and shifts from regulatory CD4+ T-cells to IFN-γ producing CD4+ T-cells and CD8+ T-cells [117]. Moreover, pro-inflammatory macrophages in adipose tissue secrete TNF-α and IL-1β. These cytokines together acts in an autocrine and paracrine manner, interferes with insulin signaling and leads to insulin resistance. Insulin resistance in adipose tissue in turn increase lipolysis rate. Similar inflammatory responses interfering with insulin signaling are also seen in other tissues, such as pancreas, liver and muscle [89]. Signaling pathways associated with hypertrophic and dysfunctional adipocytes include stress pathways like p38 and JNK that are up-regulated and activated in adipose tissue of individuals with hypertrophic adipocytes [118]. It has also been shown that activation of Ask1-MKK4-p38-JNK signaling in humans correlates with hypertrophic obesity, adipose tissue inflammation and insulin sensitivity [119].

Being a metabolically active tissue, adipose tissue synthesizes and secretes a large number of biologically active substances. The pro-inflammatory cytokines and their role in insulin resistance have been mentioned; other substances secreted from adipose tissue include leptin, resistin, adiponectin, PAI-1 and free fatty acids (FFA). FFA is released mainly due to lipolysis, an important factor in the dysfunction of adipocytes [109]. In healthy individuals, lipolysis is tightly regulated by hormonal and nutritional factors. Lipoly- sis is triggered and FFA released under conditions of negative energy balance such as fasting and during exercise. This provides sufficient supply of substrate for oxidative metabolism and is crucial for survival [120]. Insulin has anti-lipolytic effects and partly due to insulin resistance, lipolysis is typically increased in obese and T2D individuals. Increased lipolysis leads to elevated plasma FFA levels [109]. FFA is of particular interest because it fulfills all three criteria to be a physiological link between obesity and insulin resistance; it is elevated in blood of obese people, the elevation leads to increased insulin resistance and lowering of the FFA blood levels decrease insulin resistance. FFA mediates insulin resistance through inhibiting insulin signaling, thereby decreasing insulin stimulated glucose transport and/or phosphorylation [109]. FFA also activates the pro-inflammatory NF-κB pathway, which results in secretion of pro-inflammatory cytokines and chemokines [121-123]. As mentioned above, pro-inflammatory cytokines and chemokines interfere with insulin signaling and lead to insulin resistance. Taken together, obesity causes a negative spiral with hypertrophic adipocytes, inflammation and increased lipolysis that promote each other and all accelerate insulin resistance.

27 TF and FVIIa in diabetes Both T2D and obese subjects have increased plasma levels of pro- thrombotic factors, including FVII and TF procoagulant activity of circulating monocytes [124-129]. Obese T2D subjects compared with obese non- diabetics have increased plasma TF antigen and TF procoagulant activity [13]. Mice lacking PAR2 or the cytoplasmic domain of TF have been found to be protected from high-fat diet induced obesity and insulin resistance. It has also been shown in mice that TF-PAR2 signaling in hematopoietic cells promotes high-fat-diet induced adipose tissue macrophage inflammation, thereby increasing the local inflammation in adipose tissue. Moreover, studies on mice have revealed that TF/FVIIa signaling in adipocytes play a possible role in obesity regulation [117, 130].

T2D patients and obese subjects have increased expression of TF in their adipose tissue and also have increased rates of lipolysis. T2D patients have even higher levels of TF in their adipose tissue compared with their non- diabetic obese counterparts [13]. Lipolysis results in elevated circulating fatty acids that further contribute to inflammation and insulin resistance [109]. Adipose TF mRNA correlates with plasma FFA and both cytokines and FFA has proved to increase expression of TF in adipose tissue [18]. Moreover, plasma TF activity correlates with plasma FFA [13]. Since FVII is increased in plasma and TF is increased in adipose tissue of diabetic patients, it is likely that adipocytes of these individuals are subjected to TF/FVIIa signaling. The function of TF up-regulation in adipose tissue due to FFA and cytokines is not known, it might act protecting from lipolysis or be part of a viscous cycle.

28 Methods

Cell culture and isolated cells For Paper I, the adherent murine MIN6 pancreatic beta cell line was used. MIN6 were derived from a mouse insulinoma and display characteristics of pancreatic beta cells, including glucose stimulated insulin secretion. Experi- ments were also performed on isolated human islets, provided by the Uppsa- la facility for the isolation of human islets and cultured free-floating.

For paper II, many assays were performed on adherent 3T3-L1 adipocytes. 3T3-L1 is a murine fibroblast cell line that can be differentiated into adipocytes by the addition of insulin, IBMX, dexamethasone and Rosiglithazone to the media. Additional experiments were also performed on primary human adipocytes from healthy volunteers. Biopsies were obtained with needle aspiration from the subcutaneous lower abdominal depots and cells isolated by collagenase treatment.

For Paper III, the p53-mutated human MDA-MB-231 breast cancer and PC3 prostate cancer cell lines were used. MDA-MB-231 was originally established from a patient with a metastatic mammary adenocarcinoma and is an aggressive, invasive, triple-negative breast cancer. The cells have an epithelial morphoplogy, express high levels of TF, PAR1/2 and are adherent. PC3 were originally derived from a bone metastasis of a grade IV prostatic adenocarcinoma. The cells are epithelial, adherent and express moderate levels of TF and PAR1/2.

For paper IV, the human leukaemia cell line U-937 was used. U-937 was originally obtained from a pleural effusion of a patient with histocytic lym- phoma. The cells are grown in suspension and differentiated with vitamin D3 to a monocyte like phenotype. The human monocytic cell line THP-1, originally derived from an acute monocytic leukemia patient, was also used. THP-1 cells are grown in suspension and have the phenotype of human monocytes. TF expression decreases when the cells are differentiated. Mon- ocytes were isolated and purified from whole blood from healthy volunteers by standard density gradient centrifugation after negative selection (Ro- setteSep, Stem Cell Tech).

29 Protein expression and cell signaling Western blot Total protein expression and phosphorylation of proteins were investigated in cells by western blot. Phosphorylation of proteins is a way to investigate which signaling transduction pathways that are up- or down-regulated after e.g. stimulation with different reagents such as FVIIa. After culturing and treatment, cells were typically lysed in SDS sample buffer, In paper III, ly- sates were also prepared by using a Subcellular Fractionation Kit which al- lowed separate analysis of membrane, nuclear soluble and chromatin-bound cellular fractions. The samples were loaded onto SDS-PAGE gels and proteins separated based on size. Proteins were transferred to immobilon-FL PVDF membrane and subsequently incubated with primary antibodies towards the protein or phosphorylated protein of interest. The membranes were further incubated with secondary antibodies conjugated to IR-Dyes 680 or 800. Proteins were visualized with an infrared imaging system and quanti- fied with the odyssey V 3.0 software. Western blot was used in all papers of this thesis.

Flow cytometry Surface protein expression was investigated with flow cytometry. Cells were cultured, treated under different conditions, harvested, washed and incubated with FITC-labeled antibodies towards TF (Paper I and IV) or the active con- formation of ITGβ1 (paper III) with IgG as isotype control. The surface protein expression of the cells was analyzed in a FC500 flow cytometer and the mean fluorescent intensity values calculated using Kaluza software.

Protein proximity ligation (PLA) PLA is an in-situ anti-body based technique used to investigate if antibody epitopes are in close proximity to one another. Using primary antibodies towards two different epitopes of interest and adding secondary oligonucleo- tide-conjugated PLA probes, the PLA probes hybridizes if they are in close proximity (<40 nm). An amplification solution consisting of nucleotides and fluorescently labeled oligonucleotides was added together with polymerase to generate a rolling-circle amplification (RCA). The fluorescently labeled oligonucleotides hybridizes to the RCA product and is visualized by fluorescent microscopy. In situ PLA is performed on cultured cells in paper III, detecting proximity of TF and ITGβ1 and proximity of IGF-1R and Cav1 as well as tyrosine phosphorylations of IGF-1R and Cav-1.

30 Gene expression siRNA gene silencing Small interfering RNA (siRNA) transfection is a technique to silence a gene by interfering with the mRNA and thereby hinder protein translation. A siRNA complementary to a stretch of the target mRNA is transfected to the cells and incorporated into the RNA-induced silencing complex that binds the mRNA, preventing it from being translated by e.g. degradation. The effect of siRNA treatment can typically be seen on both mRNA and protein levels, since the transcribed mRNA is degraded and hence the protein never expressed. siRNA transfections were used in paper I, II and III of this thesis. qRT-PCR The gene expression of certain genes of interest was analyzed with PCR. Extracting RNA from cells and converting the mRNA to cDNA allows for analysis of expressed genes, using specific primers targeting the mRNA transcript of interest. In a similar approach, miRNA can be converted to cDNA and the expression of specific miRNAs analyzed. Total RNA was extracted using either Trizol or spin columns according to the manufacturers protocol. Purity and concentration of RNA were measured with Nanodrop. cDNA from mRNA was obtained using MMLV reverse transcriptase and oligoDT whereas miRNA cDNA was obtained using specific RT Primers and PreAmp Primers. The obtained cDNA were analyzed using real-time quantitative PCR (qRT-PCR) with specific primers for the target transcripts. miRNA were analyzed in paper IV, qRT-PCR analyzes were performed on different target genes in all papers of this thesis.

RT-PCR Reverse-transcriptase PCR (RT-PCR) analyses were performed on mRNA cDNA (obtained as described above), with Platinum Taq DNA Polymerase as described by the manufacturer, with primers targeting the transcript of interest. Samples were separated by 1% agarose gel electrophoresis and bands were visualized by gel red and UV using a Quantity One Flour-S Mul- ti-Imager. RT-PCR were used in paper II of this thesis.

31 Functional studies Apoptosis Apoptosis is tightly regulated in cells with complex signaling pathways. Activation of caspase-3 plays a central role in the execution phase of apoptosis. Two methods were used to study cell death in beta cells in paper I. In MIN6 cells, a cell death ELISA detecting enrichment of nucleosomes was used. Cell death in human islets was evaluated with staining using insulin and cleaved caspase-3 antibodies and analyzed in a microscope. Cleaved caspase-3 antibodies were also used in WB to evaluate cell death in 3T3-L1 cells treated with cytokines in paper II and in TRAIL-treated PC3 cells in paper III.

Glucose stimulated insulin secretion (GSIS) Human islets were incubated in buffer with low glucose concentration for 60 min, followed by incubation in buffer with high glucose concentration for 60 min. Media was collected and insulin concentration determined using a human insulin ELISA.

Lipolysis Lipolysis is the process where adipocytes release free fatty acid (FFA) and glycerol. Lipolysis can be stimulated with isoproterenol and is inhibited by insulin. In paper II we performed lipolysis assays on both isolated human primary adipocytes and 3T3-L1 adipocytes. The glycerol released to the medium was estimated by measuring the absorbance with colorimetric methods and using this as lipolytic index.

Human isolated adipocytes were in a 2-3% lipocrit suspension and kept in vials with Hank’s medium in a gently shaking water bath at 37°C. Cells were pre-incubated with human recombinant FVIIa (10nM, 30min). Lipolysis was evaluated at basal level and in response to isoproterenol with and without insulin pre-incubation. Glycerol released to the medium was measured by colorimetric absorbance read at 540nm with Free Glycerol reagent (Sigma) in a Tecan Magellan plate reader and used as lipolytic index. Duplicate measurements were performed for all conditions.

Lipolysis in 3T3-L1 adipocytes was performed using the Abcam lipolysis assay kit as described by the manufacturer with adjustment for volume suita- ble for 24-well plates. Briefly, cells were washed and kept in buffers sup- plied in the kit. Stimulations and conditions varied between experiments and included isoproterenol stimulation, FVIIa stimulation and TF knock-down

32 with siRNA. Glycerol released to the medium was measured by colorimetric absorbance read at 570nm.

Glucose uptake Glucose uptake was evaluated in human primary adipocytes in paper II. Iso- lated adipocytes, lipocrit 5% were suspended in glucose free medium and kept in vials in a gently shaking water bath at 37°C. Cells were pre- incubated with or without FVIIa. Cells were left untreated or incubated with insulin. D-[U-14C] glucose was added to vials and further incubated. Reac- tion was stopped by transferring adipocytes in suspension to vials on ice and adipocytes were separated by centrifugation through silicone oil. Glucose uptake was evaluated by triplicate measurements of the adipocyte-associated radioactivity in a beta-counter.

TF coagulation activity TF coagulation activity was evaluated with a FXa assay. In short, cells were incubated in HBSA with FVIIa and FX for 2h at 37°C to allow for FX to be activated. FXa formation was stopped with EDTA and chromogenic substrate added for 15min at 37°C. A standard curve was included in every experiment, ranging from 0.1pg/ml to 1000pg/ml of re-lipidated hTF. Absorb- ance was read at 405nm. All experiments were performed in duplicates. De- pending on the experiment, TF concentration was calculated based on the standard curve or relative absorbance values compared to control. Coagula- tion activity was analyzed on human isolated adipocytes as well as TF- depleted 3T3-L1 adipocytes in paper II and on differentiated U937 cells as well as isolated LPS-stimulated monocytes in paper IV.

33 Aims

Paper I The aim of paper I was to study the outcome of TF/FVIIa signaling on pancreatic beta cell viability and function. The possible effects of TF/FVIIa signaling on cytokine-induced beta cell death and islet function in vitro were investigated.

Paper II Paper II addresses the question of the functional role of TF/FVIIa signaling in adipocytes. The effects on lipolysis, glucose uptake and coagulation activity were explored.

Paper III The aim for paper III was to further study TF/FVIIa signaling with a focus to elucidate the underlying molecular mechanisms of TF/FVIIa transactivation of IGF-1R. The aim was also to investigate some functional effects of the IGF-1R induced signaling transduction pathway in model systems of PC3 prostate cancer cells and MDA breast cancer cells.

Paper IV In paper IV the aim was to investigate if miRNAs regulate the inducible TF expression in monocytic cells. TF gene and protein expression as well as activation of coagulation were used as biological readouts.

34 Results and Discussion

Paper I Background T1D and T2D are characterized by the progressive loss and failure of insulin-producing beta cells, located within the pancreatic islet of Langerhans. In T1D, the loss of beta cells is thought to result from an autoimmune like destruction of the beta cells, mediated through inflammatory cytokines such as IL-1β, TNFα and IFN-γ [100]. Islets and beta cells express TF under normal conditions and islet TF has been studied in islet transplantation, where TF mediates coagulation that leads to islet cell death after transplantation [131- 133]. Diabetic patients show increased plasma levels of TF and FVIIa and it is possible that the beta cells of these patients are subjected to TF/FVIIa signaling [14, 126]. The effect of TF/FVIIa signaling in beta cells has not been studied before. In this project we therefore investigated the viability and function of beta cells subjected to TF/FVIIa signaling in the presence and absence of cytokines.

Results and discussion The effect of cytokines on TF expression was investigated in human pancreatic islets as well as insulin producing MIN-6 cells. A combination of the cytokines IL-1β, TNF-α and IFN-γ induced TF gene- and protein expression in human pancreatic islets and MIN-6 cells, and increased the surface expression of TF in MIN-6 cells. Treating MIN-6 cells with combinations of FVIIa, cytokines, FXa and the thrombin inhibitor hirudin and measuring cell death with a cell death ELISA lead to the conclusion that FVIIa did not affect basal beta-cell death but, independently of downstream coagulation activity, augmented beta-cell death in response to cytokines (Figure 4). Treat- ment with FVIIa also increased cytokine induced cell death in human islets, measured as cleaved caspase-3 (Figure 5). The effect was proved dependent of TF, since TF down regulation abolished the effect of FVIIa stimulation. Utilizing western blot on both MIN-6 cells and human pancreatic islets, the signaling mechanism behind TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK. FVIIa addition potenti- ated cytokine-induced JNK activation and JNK inhibition abolished the

Figure 4. Cell death in MIN-6 cells. MIN-6 cells were pre-treated with FVIIa (10nM), FXa (100nM) or FVIIa + hirudin (10U/ml) for 6h, followed by treatment with a cytokine mixture of IL-1β, TNF-α and IFN-γ for 72h. Cell death was quanti- fied using cell-death ELISA by measuring absornabce (A405-A490) * = p≤0.05. Image from Edén et al, Diabetologia, 58(11) 2563-72

Figure 5. Cell death in human islets. Human islets were left untreated or treated with FVIIa (10nM) for 6h and stimulated with cytokine mixture of IL-1β, TNF-α and IFN-γ for 72h, then midly dispersed to generate single islet cells. Cells were stained with DAPI, insulin antibody and cleaved caspase-3 antibody and visualized by fluo- rescence microscopy. Graph represents number of cells (out of ca 2000/group) stained double positive for insulin and cleaved caspase-3. * = p≤0.05. Image from Edén et al, Diabetologia, 58(11) 2563-72 effect of TF/FVIIa on cytokine-induced beta cell death. On a functional level, FVIIa stimulation resulted in inhibition of GSIS from human pancreatic islets. This effect was blocked by the TF antibody 10H10 that mainly targets TF/FVIIa signaling by inhibition of PAR2 cleavage. It is therefore most possible that the effect of TF/FVIIa signaling in beta cells is PAR2 dependent.

These results indicate that TF/FVIIa signaling has a negative effect on beta cell function and promotes beta cell death in response to cytokines, inde-

36 pendent of downstream coagulation factors and possibly through PAR2 activation.

Paper II Background Dysregulation of hemostasis is linked to the development of T2D and considered a contributor to its cardiovascular comorbidities, studies have reported involvement of both coagulation and fibrinolysis pathways [18, 117, 134, 135]. Obese subjects have increased plasma concentrations of TF and FVII, increased expression of TF in their adipose tissue, and obesity has been linked to increased plasma TF activity [13, 18, 125, 136]. Moreover, TF- mediated signaling have been coupled to high-fat diet-induced obesity, adipose tissue inflammation and insulin resistance in mice [130]. Insulin has anti-lipolytic effects and partly due to insulin resistance, obese and T2D individuals have higher lipolysis rates, leading to increased FFA in plasma [109]. Plasma FFA correlates with adipose tissue TF mRNA levels that might indicate a possible role for TF in lipolysis [13].

Results and discussion First, the gene expression of TF as well as FVII was investigated in human primary adipocytes. Using RT-PCR, we found a relative strong expression of TF and a weaker but distinct expression of FVII (Figure 6). The TF expression was confirmed on protein level with western blot. 3T3-L1 adipocytes were shown to express TF on protein level, and we were able to down- regulate the TF expression using density based separation and re-plating of enriched adipocytes in a monolayer together with siRNA.

Isolated human primary adipocytes were proved coagulant active in a coagulation activity assay measuring formation of FXa. Interestingly, coagulation activity was found to be almost equally high without addition of FVIIa, indicating that the FVII produced by the cells are enough for initiation of coagulation. Addition of active site-inhibited FVIIa (FVIIai) abolished the coagulation activity. In 3T3-L1 adipocytes with siRNA down-regulated TF, the coagulation activity was suppressed with 45% (Figure 7). These results demonstrate the potency of the coagulation initiation of TF/FVIIa, where a small amount of FVIIa is enough to initiate the activity and a down regulation of TF with about 90% only result in suppression of coagulation activity with 45%.

Figure 6. mRNA expression of TF and FVII in human primary adipocytes. Human primary adipocytes were isolated from healthy volunteers. TF and FVII mRNA expression were detected with RT-PCR, with beta-actin as positive control. Adipo- cytes from all five subjects express TF and FVII on mRNA level. Image from Edén et al, Upsala Journal of Medical Sciences 124(3) 158-167

Figure 7. Coagulation activity. A) Human primary adipocytes were isolated and pre- stimulated with 100nM FVIIai for 5 min or left un-treated. Incubation was with or without 10nM FVIIa. Negative control incubated without FVIIa and FX. Graph represents absorbance values. B) TF was down-regulated using siRNA in 3T3-L1 adipocytes and coagulation activity was evaluated as described. Graph represents absorbance values as % of control. ** = p≤0.01 *** = p≤0.001. Image from Edén et al, Upsala Journal of Medical Sciences 124(3) 158-167

We found no evidence for TF/FVIIa pathway involvement in lipolysis; neither FVIIa stimulation nor TF depletion altered the lipolysis rate in any setting tested. We also failed to see any effect on lipolysis rate due to PAR1 or PAR2 stimulation. Long term (48h) cytokine stimulation increased glycerol release to the same extent as 3h of isoproterenol stimulation. FVIIa stimulation did not further alter glycerol release in response to cytokines. In our

38 experimental settings, we did not see an effect of FVIIa stimulation on glucose uptake in isolated human primary adipocytes.

The results in this study show for the first time that purified human adipocytes express TF and FVII and are coagulant active in a TF/FVIIa manner. No evidence was found for a role of TF/FVIIa signaling in neither lipolysis or glucose uptake.

Paper III Background One of the biological consequences of TF/FVIIa signaling is suppression of apoptosis by activation of PI3K/AKT pathways [9]. In an earlier study in our research group it was shown that inhibiting or removing IGF-1R strongly reduced the FVIIa-mediated phosphorylation of AKT and hence the subsequent protection against apoptosis in cancer cells [76]. The anti-apoptotic effect was found to be independent of phosphorylation of TF cytoplasmic domain, PAR1 and PAR2 signaling [76, 137]. Furthermore, FVIIai does not induce protection from apoptosis or IGF-1R transactivation [9, 137, 138]. Both TF and IGF-1R have been associated with caveolae domains [139, 140]. In this paper, we investigate the mechanism of TF/FVIIa transactivation of IGF-1R and link it to caveolae and ITGβ1.

Results and discussion PC3 cells were treated with FVIIa and the association between TF and ITGβ1 on the cell surface was investigated with PLA in situ. FVIIa treatment increased the association between TF and ITGβ1. Next, IGF-1R transactivation by FVIIa treatment in PC3 cells with siRNA knocked-down ITGβ1 was investigated. No activation of IGF-1R by FVIIa treatment was detected when ITGβ1 was depleted, compared to control cells with intact ITGβ1 were activation was recorded as expected (Figure 8). These experiments indicate that transactivation of the IGF-1R is dependent on TF/FVIIa-induced activation of ITGβ1.

Figure 8. PC3 cells were incubated with scramble or ITGβ1 siRNA for 72h and then treated with FVIIa (100nM, 30min). Phosphorylation of IGF-1R was analyzed by PLA and antibodies toward IGF-1R and phosphotyrosine (pY). * = p≤0.05 *** = p≤0.001

Caveolin 1 (Cav1) is the defining protein of caveolae and IGF-1R and Cav1 proteins were found to be in close proximity in PC3 cells, using PLA. Re- ducing Cav1 expression using siRNA lead to an increase of IGF-1R phosphorylation. Furthermore, FVIIa stimulation of PC3 cells treated with a Cav1 scaffolding domain peptide (mimicking Cav1 overexpression) failed to induce phosphorylation of IGF-1R. Taken together, it was concluded that the caveolae protein Cav1 prevented IGF-1R activation in resting cells via its scaffolding domain. FVIIa treatment did not affect protein levels of Cav1, but was found to induce Cav1 Tyr14 phosphorylation, dependent of TF and ITGβ1, but independent of PAR1/2 and IGF1. Cav1 Tyr14 has been described to be phosphorylated by kinases belonging to Abl and Src family. FVIIa treatment of PC3 cells was found to phosphorylate Src but not Abl.

PC3 cells were pre-treated with Cav1 scaffolding domain peptide and stimulated with FVIIa. Cells were fractioned and the nuclear translocation of IGF- 1R assessed by WB. FVIIa stimulation resulted in IGF-1R nuclear translocation and the effect was cancelled in presence of Cav1 scaffolding domain peptide. Challenging PC3 cells with TRAIL, we could demonstrate that the anti-apoptotic effect of FVIIa stimulation was significantly reduced when ITGβ1 was down regulated and when the Cav1 scaffolding domain was overexpressed.

In conclusion, we propose a mechanism where TF/FVIIa/ ITGβ1 signaling leads to phosphorylation of Src and subsequent phosphorylation of Cav1, independent of PAR1/2. When Cav1 is phosphorylated, its inhibitory effect on IGF-1R is cancelled, resulting in IGF-1R activation and subsequent anti- apoptotic effects via Ras/MAPK as well as nuclear translocation of IGF-1R.

40 Paper IV Background The mechanisms regulating TF gene expression on a molecular level have not been fully elucidated. The gene expression of TF is regulated by two alternative transcription binding-sites within the promoter region, the proximal promoter region regulate the constitutive expression and the distal promoter region controls the inducible expression of TF [33]. In patients with acute coronary syndrome (ACS) the levels of circulating TF are elevated for months after the first cardiac event. TF mRNA is rapidly turned over and has a half-life of 45-90 minutes, indicating that the TF protein expression in ACS and other patients with long-term elevated TF levels in the circulation must be maintained by mechanisms that have not yet been identified [141, 142]. Possible mechanisms include post-transcriptional regulation of the mRNA by small non-coding RNAs, including miRNAs [143, 144]. Some miRNAs have been shown to regulate TF expression in different cell types and cancers [7, 51, 145, 146]. In the present work we aimed to investigate the involvement of miRNA regulation of the inducible TF gene transcript, starting by screening for differentially expressed miRNAs in a cell based system were TF expression can be down-regulated. Identifying differentially expressed miRNAs, we investigated the role of one of these miRNAs; miR- 223-3p, in TF expression in monocytes.

Results and discussion The human leukaemia cell line U937 expresses TF at moderate levels, differentiation to a monocyte-like phenotype with vitamin D3 leads to down- regulation of TF gene expression. These cells, pre- and post differentiation, were used in a screen to identify differentially expressed miRNAs. One miRNA with predicted target site in the 3’UTR of the TF transcript was identified; miR-223-3p. It was demonstrated that miR-223-3p binds directly to the seed-sequence in the 3’UTR of the TF mRNA transcript. The expression of miR-223-3p was higher in differentiated compared to undifferentiat- ed U937 cells. Using qRT-PCR and investigating TF and miR-223-3p expression at different time points, 0, 24, 48 and 72h post differentiation initiation, it was shown that TF gene-expression is gradually down-regulated and miR-223-3p is upregulated over time. Hence, there is a reversed correlation between miR-223-3p gene expression and TF gene expression.

The down-regulation of TF gene expression in differentiating U937 cells over time were confirmed on protein level at the same timepoints, using western blot and flow cytometry. Furthermore, a concordant down-

41 regulation of TF coagulation activity was also seen at the different time points.

Purified monocytes from healthy volunteers were induced to express TF by stimulation with LPS and the gene expression of TF and miR-223-3p were monitored. The results show that as TF expression increases with LPS stimulation, the miR-223-3p expression decreases.

Based on these results, it was concluded that when miR-223-3p increases, TF expression decreases, both on the gene- and protein levels as well as the coagulation activity. The opposite was also found to be true, when TF is induced, the miR-223-3p expression is reduced. Combined, these results show that miR-223-3p regulates the human TF mRNA transcript post- transcriptionally in human monocytes.

42 General discussion and future perspectives

TF is a vital molecule and the absence of TF is not compatible with life. TF is expressed on numerous cell types, mainly outside the circulation where it acts as a hemostatic envelope and initiates the coagulation in case of tissue damage. Apart from coagulation, TF has also proved to act as a true signaling molecule and play important roles in e.g. migration and apoptosis of cancer cells and diet-induced obesity. The overall aim of this thesis was to study the regulation of TF, in depth downstream signaling, and the functional role of TF expression in different cell types, apart from coagulation. Moreover, TF has been implied to play a role in diabetes, a disease affecting millions of people worldwide. This thesis aimed to elucidate some functions of TF and TF/FVIIa signaling in cells of importance in diabetes, i.e. beta cells and adipocytes.

The regulation of TF by miRNAs and the functional role thereof were studied in monocytes. The TF expression decreases over time in U937 cells undergoing differentiation to a more monocyte like phenotype. In a screen for differentially expressed miRNAs in these cells undergoing differentiation, one miRNA with predicted binding site at the 3’ UTR of TF mRNA was identified; miR-223-3-p. Further experiments revealed that miR-223-3p increases as TF expression decreases both on gene- and protein level in these cells. The decrease of TF was reflected in a reduction of coagulation activity. In purified human monocytes induced to express TF with LPS it was evident that miR-223-3p decreases when TF expression increases. It was concluded that miR-223-3p regulates the inducible TF expression in monocytes and this is reflected in the coagulation activity.

TF/FVIIa signaling has anti-apoptotic effects in models of prostate and breast cancer. Using PC3 prostate and MDA-MB-231 breast cancer cells as models, the signaling transduction pathway down stream TF/FVIIa leading to protection from TRAIL induced apoptosis was studied in depth. Previous studies have revealed a trans-activation of IGF-1R leading to subsequent anti-apoptotic effects as well as translocation of the receptor to the nucleus. The transactivation of IGF-1R was found to be dependent on ITGβ1, phosphorylation of src and Cav1.

43 The expression and functional role of TF and TF/FVIIa signaling was studied in cells of importance in diabetes. In the insulin producing beta cells it was shown that TF expression is increased by stimulation with cytokines. TF/FVIIa signaling leads to increase of apoptosis induced by cytokines in beta cells. On a functional level, it was also shown that isolated human pancreatic islets subjected to TF/FVIIa signaling release less insulin in response to glucose compared to control islets. Investigating the expression and function of TF and FVIIa in adipocytes, it was evident that the cells express TF that is coagulant active and also that isolated human primary adipocytes produce sufficient amounts of FVII to initiate the coagulation. We found no evidence for a role of TF/FVIIa in lipolysis or glucose uptake in the adipocytes.

TF/FVIIa in diabetes Both T1D and T2D are characterized by high blood glucose levels due to cells failing to take up glucose, but the cause and development of disease are distinctly different. Put simply, in T1D there is an auto-immune destruction of beta cells from invading immune cells. T2D is classically seen as a lifestyle disease with obesity and the metabolic syndrome as explanatory models for development of disease. Interestingly, both T1D and T2D patients display increased levels of TF and FVII in their blood stream and have increased risk of thrombus. The cause of this, and the actions of TF/FVIIa are still largely unknown. In Paper I, the role of TF/FVIIa in beta cells are examined and the results show that TF is up-regulated by cytokines and that TF/FVIIa signaling via JNK phosphorylation in addition to cytokines cause beta cell death. This was found to be independent of downstream coagulation. On a functional level, TF/FVIIa signaling impairs glucose stimulated insulin secretion (GSIS) from human pancreatic islets. In these experiments, the TF antibody 10H10 that mainly targets TF/FVIIa signaling by inhibition of PAR2 cleavage was used, indicating that the effects of TF/FVIIa on GSIS is likely to be PAR2 dependent. This is line with previous studies in MIN-6 cells where PAR2 activation led to inhibition of GSIS [147, 148]. Cytokines are released by immune cells and inflammatory destruction of beta cells are a hallmark of T1D. As mentioned, the metabolic syndrome is an explanatory model for T2D, and is characterized by a low-grade systemic inflammation. Hence, one common denominator in development of both T1D and T2D is inflammation. We have shown that cytokines up-regulate TF in beta cells and an increased inflammatory burden could be part of the explanation for the increased levels of TF and FVII seen in both T1D and T2D. In a study in mice linking TF/FVIIa signaling with T2D, adipose tissue and inflammation it is shown that TF/FVIIa signaling promotes high-fat-diet-induced obesity, adipose tissue inflammation and insulin resistance [130].

44 In paper II, we investigate adipocytes as they are of importance in the development of T2D and have been coupled to TF/FVIIa signaling. The results in paper II show that human adipocytes express TF and produce enough FVII to initiate coagulation but we found no evidence for a role of TF/FVIIa in either lipolysis or glucose uptake. There are many studies that have pointed to an association between adipose tissue and hypercoagulability [149] and the present study add to this by providing evidence for both isolated human primary adipocytes and murine adipocytes to be coagulant active. In paper II, adipocytes from the subcutaneous adipose tissue (SAT) were isolated from healthy volunteers. SAT is associated with venous thrombosis and shows stronger relationship with functional measurements of hypercoagulability after injury compared to visceral adipose tissue (VAT) [150]. In a study comparing T2D with non-diabetics it was found that AT-III is increased in VAT from T2D subjects [151]. SAT and VAT have distinct characteristics but it can be speculated that AT-III could be increased in SAT of T2D subjects as well. qRT-PCR analysis of TFPI and AT-III in the isolated human primary adipocytes revealed that these are expressed in the adipocytes, which most likely explain why there are not even more clinical events in the form of thrombosis. In the present study, adipocytes was only isolated from healthy volunteers. It would be interesting to investigate the expression of TF, FVII, AT-III and TFPI in adipocytes from the subcutaneous depots of both healthy volunteers and T2D subjects.

Lipolysis experiments were done with cytokines in the 3T3-L1 adipocytes, with addition of FVIIa. Cytokine treatment did increase glycerol release but no additional effect of FVIIa stimulation and no phosphorylation of hormone sensitive lipase (HSL) were detected. The effect on glycerol release was instead most likely due to apoptosis since an increase in cleaved caspase 3 was detected. However, we have not fully investigated the possible combinatory effects of cytokines and TF/FVIIa signaling in adipocytes and previous studies have reported that IL-1β and TNF-α can induce lipolysis and that TNF-α contributes to insulin resistance in adipose tissue [152]. Moreover, adipocytes are not the only peripheral cell type affected by insulin resistance. It would be of interest to investigate the possible role of TF/FVIIa on glucose up-take in e.g. muscle cells and examine the combinatory effect of low- grade inflammation. Taken together, our results show a specific role of TF/FVIIa in beta cells where they augment cytokine induced beta cell death and impairs insulin secretion, but although present in adipocytes does not seem to effect lipolysis or glucose uptake.

45 The potency of TF/FVIIa coagulation initiation It is known that the activation of FX to FXa is initiated by picomolar concentrations of FVIIa and that only a few TF receptors on the cell surface are needed for this initiation. Several of our experiments underline this fact. Some cancer cells/tumors are known to produce FVII, which can result in TF/FVIIa/PAR2 signaling and subsequent events without the requirement of FVII from circulation [153]. In paper II we show that human primary adipocytes produce their own FVII and that the produced amount is enough to initiate formation of FXa. Using RT-PCR we detect bands of FVII in purified adipocytes from five different donors. The coagulation assay proves that the amount of FVII produced by the human primary adipocytes are biological relevant. In 3T3-L1 adipocytes we used siRNA to down-regulate TF and consistently rendered a knock-down of around 90%. Despite this 90% knock-down of TF, coagulation activity was only dampened with some 45%. Moreover, in Paper IV, U937 cells are differentiated to monocytes and using both WB and flow cytometry it is evident that the TF protein expression are reduced with up to 70% over time. This reduction is reflected in the coagulation activity assay with no more than a 30% reduction of coagulation activity. Taken together, these results demonstrates the power of TF/FVIIa coagulation initiation, were small amounts of FVIIa and few TF receptors are needed to generate a significant formation of FXa.

The complexity of TF/FVIIa intracellular signaling transduction In paper III the pathway of TF/FVIIa transactivation of the RTK IGF-1R and subsequent anti-apoptotic effect was investigated in depth in PC3 prostate- and MDA-MB-231 breast cancer cells. TF/FVIIa is known to transactivate the RTKs EGFR and PDGFRβ through Src-dependent mechanisms [74, 75] and this was found to be true also for transactivation of IGF-1R. In the present study, the Src phosphorylation was conveyed through ITGβ1, which has not been reported for transactivation by TF/FVIIa of the other RTKs. How- ever, interplay between TF and the integrin subunits β1, α3 and α6 has been reported elsewhere [81]. In line with previous publications from our research group and others, the formation of TF/FVIIa/ITGβ1 complex is dependent on FVIIa, but not on phosphorylation of TF cytoplasmic domain or PAR2 activation [76, 82, 137]. Moreover, the IGF-1R transactivation was found to be dependent on Cav1 phosphorylation. There are conflicting results published on the relation of IGF signaling and Cav1 [154-156]. The outcome of protein interactions with Cav1 seems to be dependent on cell type, protein and type of stimuli. However, TF dependent phosphorylation of Cav1 by FVIIa was shown in three different cell lines in paper III so the connection

46 cannot be disregarded as exclusive to PC3 cells. In the context of insulin receptor (IR) signaling, IGF-1R has overlapping functions with IR. Both IR and IGF-1R regulate metabolism and gene expression through the major pathways PI3K/AKT and Ras/MAPK. Moreover, IGFRs and IR can form heterodimers [157]. It has even been proposed that IR and IGFR act as identical portals for gene expression regulation, and that the specific ligand- receptor interaction results in modulation of signal amplitude thereby causing the differences between insulin and IGF1 effects [158]. However, the anti-apoptotic effect by TF/FVIIa in TRAIL treated PC3 and MDA-MB-231 cells is dependent on IGF-1R and IR is unlikely to be involved. siRNA knock-down of IGF-1R as well as blocking IGF-1R with the inhibitors AG1024 or picropodophyllin resulted in FVIIa failing to rescue the cells from the induced apoptosis [76]. The possible interaction between TF/FVIIa and IR has not been fully investigated.

Regarding TF/FVIIa signaling and apoptosis, there is opposite effects in beta cells and in cancer cells. In paper I, we show that TF/FVIIa signaling increase the cytokine induced beta cell death, whereas in paper III there is an anti-apoptotic effect of TF/FVIIa signaling. Anti-apoptotic signaling of TF/FVIIa was first reported in 2003 [9] and later publications have also further documented the effect in TRAIL-receptor activated PC3 prostate and MDA-MB-231 breast cancer cells [137]. In contrast, and in line with our findings in paper I, it was recently reported that TF has pro-apoptotic effects in endothelial cells in response to MAPK activation [159]. This is of course intriguing and since there is a lack of perfect explanatory models, only spec- ulations can be made. First and foremost, the experiments are done in different cells. Different cells have different characteristics and different receptors on their cell-surfaces. Moreover, the experimental settings are different. Cy- tokines are used to challenge the beta cells, and the apoptotic effect of TF/FVIIa is only seen in addition to cytokines. Two common denominators can be pointed out between our results from paper I and the study by Ethaeb et. al. [159] where TF signaling show pro-apoptotic effects; they are done in non-malignant cells and the effect is seen after MAPK activation. In fact, beta cells treated with TF siRNA showed a small increase in cell death compared with control cells under basal conditions. The argument is made that TF might promote survival of unstressed beta cells but mediate cell death in response to inflammatory stress. In paper III we induce apoptosis in PC3 cells with TRAIL. One interesting experiment would be to challenge PC3 with cytokines and likewise induce apoptosis with TRAIL in beta cells and investigate how TF/FVIIa affect the outcome. The investigated pathways in paper I where those known to be activated by cytokines, and it was found that JNK activation mediate the effect. We also argue that the signaling is PAR2 dependent, due the effect of the TF antibody 10H10 on GSIS. Even though TF/FVIIa signaling is involved in both paper I and III, we are look-

47 ing at different pathways and, in contrast to paper I, the signaling in paper III seem to be PAR2 independent.

When it comes to intracellular signaling, one must keep in mind that the existing explanatory models and the conclusions drawn from experiments is likely to be far from a more complex reality. Experiments done in vitro are simplified models with well-defined cells in a controlled environment. An in vivo setting is far more complex than what can be investigated in vitro. A strength in this thesis is that human cells have been used in most experiments. In paper I we use the murine MIN-6 beta cells but the results are confirmed in isolated human pancreatic islet. Likewise, in paper II we use murine 3T3-L1 cells but when possible, the results are confirmed in isolated human primary adipocytes. miRNA In paper IV, we examine the regulation of TF by miRNA in monocytes. The first miRNA was discovered in the early 1990s [160] and miRNAs was rec- ognized as a distinct class in the early 2000s [161]. In the last 10 years the research on miRNA and their possible use as biomarkers, targets or therapeutic agents in a wide range of diseases has increased markedly, including cardiovascular diseases [162]. TF is a potent trigger of coagulation and has been shown to be up-regulated in diseases such as cancer, diabetes and in cardiac patients, causing complications such as thrombus formation. miR- NAs involved in TF regulation could therefore be a potential therapeutic target or agent in these settings. Several miRNAs have been shown to directly regulate TF protein expression in different cell types and tissues [7] and have implications in a variety of different diseases, including the ones mentioned above. miR-19a has been shown to suppress TF expression in colon cancer cells, human microvascular endothelial cells and in human monocyte THP-1 cells [50, 145]. miRNA regulation of TF has been reported to contribute to the complications and pathogenesis of diabetes. miR-19a and miR- 126 exhibits antithrombotic properties by targeting vascular TF expression and thereby impacting the hemostatic balance of the vasculature in diabetes mellitus [51, 145]. miR-19b and miR-20a reduce TF expression and procoagulant activity in vitro. Monocytes from patients with systemic lupus erythematosus and antiphospholipid syndrome have lower levels of miR-19b and miR-20a as compared to healthy subjects [48]. In paper IV, the TF regulation by miR-223-3p in monocytes is investigated. miR-223-3p has previous been shown to be down-regulated in vascular endothelial cells in mice when TF expression was induced using TNF-α [163]. The examples above underline some of the complexity of miRNAs, one miRNA can have a multi- tude of targets and several miRNAs can act on the same target gene.

Considering how relatively new the field of miRNA research is and the amount of processes it is involved in, we have likely only seen the beginning of a new and interesting field of research, where our study add a small piece to the puzzle.

49 Conclusions

• TF expression in human monocytes is regulated by miR-223-3p.

• miR-223-3p play a role in regulating the inducible TF expression but can also effect the constitutive TF expression in experimental settings in vitro.

• The IGF-1R transactivation due to TF/FVIIa signaling is dependent on the cytoplasmic domain of TF, ITGβ1, Cav1 and Src-activation but independent of PAR2.

• The transactivation of IGF-1R by the TF/FVIIa complex leads to IGF- 1R translocation to nucleus and anti-apoptotic effects in cancer cells.

• TF/FVIIa signaling augments cytokine induced beta cell death.

• The apoptotic TF/FVIIa signaling in beta cells is dependent on JNK activation and is likely PAR2-dependent.

• No role was found for TF/FVIIa in lipolysis or glucose uptake in adipocytes.

• Isolated primary human adipocytes express coagulant active TF.

• Human adipocytes produce FVII in an amount that renders TF/FVIIa coagulation activation.

50 Populärvetenskaplig sammanfattning på svenska

Alla har vi någon gång haft ett sår som blöder och utan att egentligen fun- dera på det en stund senare konstaterat att det slutat blöda och bildats en skorpa. I dagligt tal säger man att blodet levrar sig, med medicinska termer säger man att blodet koagulerar. Det här styrs av en mängd olika faktorer och balansen mellan dem är livsviktig för oss. Vid en skada måste blodet koagulera för att vi inte ska förblöda. Om blodet istället koagulerar när det inte behövs riskerar man att få en blodpropp, vilket också kan vara livsho- tande.

Två faktorer som styr blodets koagulation är vävnadsfaktorn (fortsatt kallat TF, efter dess engelska namn tissue factor) och faktor VIIa (som vi förkortar FVIIa). TF är en receptor som sitter fast på ytan av celler och när FVIIa binder till TF bildas det s.k. binära komplexet TF/FVIIa och koagulationen startar. TF sitter på celler som inte har direktkontakt med blodet. FVIIa tillverkas främst i levern, frisätts till blodet och transporteras runt i blodkärlen. Tack vare att TF finns utanför blodkärlen medan FVIIa transporteras i blod- kärlen hålls de i normala fall åtskilda och blodet koagulerar inte. Om du däremot får en skada på ett blodkärl så kommer FVIIa åt att binda in till TF och blodet i området närmast skadan börjar då koagulera och på så sätt stop- pas blödningen. Tidigare har forskning visat att vissa sjukdomstillstånd är förknippade med ökad mängd av TF och FVIIa och även med en ökad risk för att få blodproppar. Det här gäller bl.a. diabetes och vissa former av cancer.

Utöver att initiera, starta, koagulationen kan TF/FVIIa även bidra till signalering in till cellen som TF sitter på. Tidigare studier i bl.a. vår forskargrupp har visat att den här signaleringen t.ex. kan bidra till cellers överlevnad eller att de migrerar (flyttar på sig). I den här avhandlingen undersöks betydelsen av TF/FVIIa signalering vid diabetes och cancer. Främst tittar vi på den signalering som inte är kopplad till koagulationen. I arbete I och II undersöks hur celltyper som är involverade i diabetes påverkas av TF/FVIIa signalering. I arbete III undersöker vi i detalj en signalväg i cancerceller där TF/FVIIa signalering hjälper cancercellen att överleva. I det fjärde arbetet

51 tittar vi istället på hur tillverkningen av TF regleras inne i cellen med hjälp av något som heter mikro RNA.

Arbete I: När vi äter bryts maten ner till glukos som tas upp från mag- tarm- kanalen till blodet och blodsockret höjs. När blodsockret höjs frisätts insulin som hjälper kroppens celler att ta upp glukos och blodsockret sänks igen. Insulinet produceras och frisätts från betaceller som finns i bukspottkörteln. Om man har diabetes innebär det att man har förhöjt blodsocker för att cellerna inte tar upp glukos från blodet. Det kan bero på att betacellerna är ska- dade och inte frisätter insulin som de borde. En sak som man vet kan skada och döda betacellerna är inflammation. Vi har undersökt hur betaceller på- verkas av TF/FVIIa signalering när de samtidigt utsätts för inflammation. I våra experiment har vi odlat betaceller i laboratoriet och behandlat dem på olika sätt för att se hur de reagerar. Det vi såg i våra experiment är att om vi slår på TF/FVIIa signalering i betaceller som samtidigt är utsatta för inflammation så ökar celldöden. Vi såg också att TF/FVIIa signalering försäm- rar cellernas förmåga att frisätta insulin. Våra försök tyder därmed på att TF/FVIIa signalering skadar betacellerna och hämmar frisättning av insulin från dem.

Arbete II: En celltyp som är intressant vid diabetes är fettcellerna. Fettcel- lens funktion är att fungera som en energireserv, när det inte finns tillräckligt med energi i form av glukos så frisätter fettcellerna fett i form av fettsyror till blodet. Kroppens andra celler kan då använda sig av fett som energikälla istället för glukos. När man har diabetes klarar inte kroppens celler av att ta upp socker från blodet, vilket kroppen tolkar som att det är brist på energi och då frisätts fettsyror från fettcellerna, i en process som kallas lipolys. Precis som andra celler tar fettcellerna normalt sett upp glukos från blodet. I det andra arbetet i den här avhandlingen har vi studerat vad TF/FVIIa har för roll i fettceller. Våra experiment visade att fettcellerna har TF och att de också producerar FVIIa. Vi såg att TF/FVIIa är koagulationsaktiva i fettcellerna och att fettcellerna själva tillverkar FVIIa som kan starta koagulationen. Vi kunde inte se att TF/FVIIa spelar någon roll för vare sig lipolysen eller glukosupptaget i fettcellerna.

Vår forskning i arbete I och II bidrar med ny kunskap om TF/FVIIa:s roll vid diabetes. Ju mer vi förstår om de molekylära mekanismerna bakom en sjuk- dom, desto större chans finns det att hitta nya sätt att bota eller lindra sjuk- domen.

Arbete III: Mekanismerna bakom när en cell ska dela sig, flytta på sig eller dö är hårt kontrollerade. En cancercell är egentligen en helt vanlig cell där den här kontrollen har slutat fungera så att cellen överlever, delar sig och flyttar sig på ett sätt som är farligt för oss. Det här leder till att tumörer och

52 metastaser bildas. För att förstå hur cancer uppstår och upprätthålls och hur vi ska kunna förhindra eller bota det, måste vi förstå vad som leder till att kontrollen i cellen inte fungerar. Vad som händer inne i cellen styrs till stor del av olika signaltransduktionsvägar.

En signaltransduktionsväg i en cell kan liknas vid en kedjereaktion. Ett ämne binder in till en receptor på cellens yta som reagerar och signalerar in till cellen och en kedjereaktion startar. En signaltransduktionsväg kan bestå av väldigt många led och involvera flera andra receptorer på cellens yta som slås på, eller av, innan det till sist leder till sista steget i signaleringen och cellen svarar med en biologisk funktion. Den biologiska funktionen kan t.ex. vara att cellen överlever, flyttar på sig eller tillverkar mer proteiner. I arbete tre har vi undersökt en signaltransduktionsväg i prostata- och bröstcancer celler som börjar med att FVIIa binder in till TF och leder till den biologiska funktionen att cellen överlever. Starten och den biologiska funktionen på den här signaltransduktionsvägen var känt sedan tidigare, vårt arbete har bidragit till att kartlägga vad som händer däremellan. På lång sikt kan den kunskap vi bidragit med vara en del i att hitta nya botemedel mot cancer.

Arbete IV: Kroppen är uppbyggd av celler som ser olika ut och fungerar på olika sätt. Det som till stor del styr hur cellerna fungerar och ser ut är våra gener. En gen kan liknas vid en kod med information och finns lagrad i DNA i kärnan av våra celler. Alla celler i kroppen har samma DNA. Det som gör att cellerna ser ut och fungerar på olika sätt är vilka gener, vilken information, som läses av och uttrycks. När en gen uttrycks läses DNAt av och ett RNA tillverkas. Om genen som uttrycks är kod för ett protein så tillverkas messenger RNA (mRNA) och man pratar om genuttryck eller uttryck på gennivå. mRNA i sig är också en kod och när den läses av tillverkas protein och man pratar då om proteinuttryck eller uttryck på proteinnivå. Vilka gener som uttrycks regleras på flera sätt, både på gennivå och proteinnivå. TF är ett protein och uttrycks först som mRNA och sedan som protein.

Ett sätt som cellen kan reglera proteinuttryck på är med hjälp av något som kallas mikro RNA. Precis som när mRNA tillverkas så bildas mikro RNA när dess gen läses av från DNAt i cellkärnan. Precis som namnet låter så är mikro RNA väldigt små former av RNA. Micro RNA är inte en kod för protein, utan dess funktion är istället att hindra att proteiner uttrycks från mRNA. Det gör de genom att binda in till mRNA och därmed stoppa dess kod från att läsas av och då uttrycks inte proteinet. I det fjärde arbetet har vi undersökt hur mikro RNA reglerar proteinuttrycket av TF.

I våra experiment undersökte vi hur mikro RNA och TF uttrycks i monocyter. Monocyter är en vit blodkropp som finns i blodet och bidrar till vårt immunförsvar. Monocyter har väldigt lite TF på ytan, men i laboratoriet kan

53 vi stimulera monocyter så att de producerar mer TF. Det vi upptäckte i det här arbetet är att ett specifikt mikro RNA, miR-223-3p, styr uttrycket av TF. I normala fall finns det ganska mycket miR-223-3p i monocyterna som då hindrar att TF mRNA koden läses av och på så sätt blockerar att TF proteinet uttrycks. När vi stimulerar cellen till att uttrycka mer TF så minskar istället miR-223-3p och TF kan uttryckas.

Vår forskning bidrar till att visa hur ett specifikt mikro RNA styr uttrycket av TF. Vid vissa sjukdomstillstånd ökar uttrycket av TF och bidrar till ökad risk för t.ex. blodproppar. Mikro RNA skulle eventuellt kunna fungera som ett läkemedel för att kontrollera att uttrycket av TF inte ökar.

54 Acknowledgments

Jag vill börja med med att tacka mina handledare och kollegor:

Min huvudhandledare, Agneta Siegbahn, din strålande expertis och långa erfarenhet har varit en enorm tillgång. Du har varit en tillsynes aldrig si- nande källa till idéer, nya infallsvinklar och inspiration. Tack för att du så entusiastiskt har delat med dig av din kunskap!

Min bihandledare Mikael Åberg som har fler signaltransduktionsvägar me- morerade än vad som borde vara möjligt. Tack för att du tog dig an mig när jag kom tillbaka och för att du på ett pedagogiskt sätt har gjort ditt absolut bästa för att lära mig allt jag borde veta.

Min bihandledare Dariush Mokhtari, tack för allt du lärt mig men framför- allt för att du gav mig chansen att doktorera.

Alla nuvarande och tidigare medarbetare i koagulation och inflammations gruppen; Lena Kask och Åsa Thulin, det är för mig en gåta att labbet hållits flytande utan er. Tack för allt stöd både i och utanför labbet och för alla fina löpturer. Jenny Alfredsson the awesome boss lady, tack för fint samarbete på manus IV. Helena Vretman det har varit bra mycket tråkigare utan dig! Oskar Eriksson, som visade hur det går till att doktorera och vars avhand- ling jag lusläst. Christina Christersson som då och då susar förbi med glada tillrop. Avinash, som varit ett välbehövligt tillskott och en utmärkt kontorskamrat.

Alla på klinisk diabetologi och metabolism: Jan Eriksson och Maria Pe- reira, tack för fint samarbete! Tack även till Cherno, Prasad, Gretha, Greg och förstås min BFC Assel.

Tack till alla kollegor på klin kem, nuvarande och tidigare, som gjort att det varit roligt att gå till jobbet även de gånger när jobbet inte varit roligt: Sofia Holmgren för alla getter och anekdoter. Evje för alla gånger jag skrattat så jag gråtit. Jenny Rubin för alla uppstyrda firanden och fredagskakor. Mia Lampinen för att du är en sådan inspiration (och i smyg min idol). Johan Stålberg, jag förväntar mig att du kör gräsklipparen på dispfesten. Lena

55 Moberg och Karin Eriksson som i det närmaste kan beskrivas som korrido- rens stöttepelare. Louise Dubois, som länge var min enda doktorandkollega. Marie Carlsson för att du stöttat och hejat på.

Ett stort tack går till alla de som kanske inte alls vet vad jag gjort på jobbet men som varit ovärderliga i allt det andra. Utan er hade jag inte klarat av de senaste åren med sinnet i behåll. Till de som förgyller min fritid och gör livet till en alltigenom trevligare upplevelse:

Maggie och Linda, för att ni finns där när det verkligen gäller, för alla djupa diskussioner, allt stöd och alla skratt.

Minas, Elin, Gustav, Sara och Axel, biomedicin-versionen av the big bang theory. Tack för alla diskussioner om stort och smått, men framförallt för alla gånger jag fått träningsvärk i magen efter att ha skrattat en hel kväll. “Den som håller kudden får prata!”

Fallskärmshopparna; Nisse, Smulan, Lisa m.fl. för alla galna upptåg och oförglömliga stunder, för att ni är de ni är och låter mig vara den jag är.

Klättrarna; Adam, Rai och Erik m.fl. för att ha pushat mig mot högre höj- der och samtidigt sett till att jag håller mig ödmjuk. “Du försökte ju inte ens!”

Dansarna; Sebastian, Laura, Diego, Frida, m.fl. som tillsammans utgör en kreativ ventil och en effektiv stress-relief. “Vamonos muchachos!”

Till min närmaste familj räcker orden egentligen inte alls till, men i alla fall:

Mormor; tack för alla luncher, middagar, fikastunder, diskussioner och re- sor. Du är och förblir en av mina stora förebilder.

Storebror Robert; min första och största idol. Äpplena föll kanske inte långt från träden och tydligen inte långt från varandra heller. Syster yster har alltid sneglat mot vad storebror gör och sen försökt göra tvärtom i trots, bara för att landa precis bredvid.

Mamma Leila och pappa Jan; tack för er villkorslösa kärlek och ert stöd, genom allt. Tack för att ni visat mig vilka värderingar och vad som verkligen är viktigt i livet.

56 References

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65 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1626 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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