Expression of interleukin 6 is modulated by prostaglandine receptor EP4

Vezava antigena na imunoglobulinski receptor BCR izzove aktivacijo in proliferacijo pri zrelih limfocitih B ter apoptozo pri nezrelih limfocitih B. Stimulacija BCR aktivira signalne poti v celici, ki vodijo do transkripcijskega reprogramiranja. Poveča se tudi izraţanje gena Ptger4, ki kodira prostaglandinski receptor EP4, na katerega se veţe naravni ligand prostaglandin E2. Prostaglandini so ţe dlje časa znani kot modulatorji imunskega odziva. Vezava prostaglandina E2 na receptor EP4 povzroči aktivacijo od cikličnega AMP odvisnih in neodvisnih signalnih poti, ki vodijo v modulacijo BCR induciranega fenotipskega odziva limfocitov B. Aktivacija BCR inducira izraţanje gena za interlevkin 6, ki ima v svoji promotorski regiji vezavno mesto za transkripcijski faktor CREB. Namen diplomskega dela je pojasniti vlogo receptorja EP4 pri modulaciji BCR induciranega izraţanja interlevkina 6. Pri eksperimentalnem delu smo se posluţili farmakološkega pristopa. Celično linijo WEHI 231 smo stimulirali z različnimi agonisti, antagonisti in inhibitorji signalnih poti. Vpliv stimulacije receptorja EP4 na BCR inducirano izraţanje gena za IL-6 smo spremljali z metodo veriţne reakcije s polimerazo v realnem času, izločanje IL-6 v celični medij pa s citometričnim citokinskim testom. Z encimsko imunskim testom za cAMP smo ugotavljali z receptorjem EP4 posredovano nastajanje cikličnega AMP v WEHI 231 celicah. Vlogo EP4/cAMP odvisnih signalnih poti pri modulaciji BCR induciranega izraţanja IL-6 smo ugotavljali z uporabo izbranih inhibitorjev encima adenilat ciklaze in protein kinaze A. V zadnjem delu diplomske naloge smo še določili vpliv IL-6 na proliferacijo WEHI 231 celične linije z metodo MTS. Ugotovili smo, da stimulacija WEHI 231 celic s PGE2 poviša z BCR inducirano izraţanje gena za IL-6. Z uporabo specifičnega agonista in antagonista receptorja EP4 smo dokazali, da so učinki PGE2 posredovani preko EP4 podtipa receptorja. Ker stimulacija receptorja EP4 sama po sebi nima vpliva na izraţanje gena za IL-6, sklepamo, da stimulacija receptorja EP4 modulira BCR inducirane signalne poti. Stimulacija receptorja EP4 je povzročila dvig intracelularne koncentracije cAMP. Z inhibicijo encima adenilat ciklaze in encima protein kinaze A smo dosegli popolno inhibicijo z receptorjem EP4 posredovane modulacije BCR induciranega izraţanja in izločanja IL-6. S tem smo dokazali, da poteka modulacija BCR induciranega izraţanja in izločanja IL-6 v nezrelih limfocitih B preko cAMP/PKA odvisne signalne poti, ki najverjetneje vodi v povečano aktivacijo transkripcijskega faktorja CREB. Izločeni IL-6 deluje na celično linijo WEHI 231 stimulatorno, saj se je proliferacija celic v prisotnosti rekombinantnega IL-6 povečala. Kljub povečanemu izraţanju gena za IL-6 ob hkratni stimulaciji receptorja EP4 in BCR, pa stimulacija obeh receptorjev vodi v zniţano izraţanje gena za receptor za IL-6, kar nakazuje obstoj negativne povratne zanke.Ligation of antigen to immunoglobulin receptor BCR induces activation and proliferation of mature B cells and apoptosis of immature B cells. Stimulation of BCR activates signal pathways in cells that induce transcriptional reprogramming. One of the genes, whose expression is highly induced, is also Ptger4, that codes for prostaglandin receptor EP4. Endogenous ligand for this receptor, PGE2, is a well-known immunomodulator. Ligation of PGE2 on EP4 receptor activates cAMP-dependent and independent pathways that promote modulation of BCR induced phenotypic response of B lymphocyte. Activation of BCR induces interleukin 6 gene expression, which has CREB binding site in promotor region of the gene. We investigated the role of receptor EP4 in modulation of BCR induced interleukin 6 gene expression. The experimental work was based on pharmacological treatment of WEHI 231 cell line. We stimulated the cells with agonists, antagonists and inhibitors of signal pathway components. Measuring the interleukin 6 gene expression after stimulation of EP4 receptor and BCR helped us to determine how both receptors modulate the expression of the fore mentioned gene. Besides that we also measured the excretion of interleukin 6 to cell media with cytometric cytokine assay. Using the enzyme-linked immunoassay we measured the concentration of cAMP after ligation of EP4 receptor. The function of EP4/cAMP dependent signal pathways in modulation of BCR induced interleukin 6 gene expression was determined by using relevant inhibitors of adenylate cyclase and protein kinase A. In the last part of our research work we determined the effect of interleukin 6 on proliferation of WEHI 231 cell line using the MTS method. The results clearly show that stimulation of WEHI 231 cells with PGE2 raises BCR induced interleukin 6 gene expression. Using specific EP4 receptor agonist and antagonist we proved, that PGE2 mediates its effects mainly through EP4 receptor. As ligation of EP4 receptor without simultaneous ligation of BCR does not induce interleukin 6 gene expression, we conclude, that ligation of EP4 receptor modulates BCR induced interleukin 6 gene expression. The stimulation of EP4 receptor raised intracellular production of cAMP. By inhibiting the enzyme adenylate cyclase and protein kinase A we managed to completely diminish the effects of EP4 receptor stimulation on BCR induced interleukin 6 gene expression. We summarize, that stimulation of EP4 receptor modulates BCR induced interleukin 6 gene expression via cAMP/PKA dependent signal pathway. Most probably this pathway leads to activation of CREB nuclear factor. Excreted interleukin 6 stimulates proliferation of WEHI 231 cells. Despite the rise in interleukin 6 gene expression after simultaneous stimulation of EP4 receptor and BCR (as we proved in the first part of our research) the expression of interleukin 6 receptor gene remains low. Even more, its expression reduces 4 hours after stimulation of EP4 receptor and BCR which could indicate a negative feedback loop

A computational and experimental study of protein localisation determinants in the mammalian endomembrane system

The subcellular localisation of a protein, together with its sequence and structure, provide the first information about its function. Although several approaches for determining localisation exist, the most widely used experimental methods involve the overexpression of a GFP-tagged construct of the protein in a cultured cell or staining the endogenous protein with specific fluorescently-labelled antibodies. Computational sequence-based localisation predictors have been developed, and are of value, but they are still limited in their predictive power. Considering the now extensive use of imaging approaches, it is therefore unsurprising that much effort has been put into the development of automated image analysis methods to classify localisation. Image classifiers are typically trained on ground truth data, which introduces certain bias. The aim of modern algorithms is to eliminate human interaction and instead perform unsupervised classification of the images. The study presented in this thesis addresses three aspects of protein localisation methodology: sequence-based localisation prediction with short linear motifs (SLiMs), unsupervised image analysis with texture features and experimental determination of protein localisation.SLiMs are 3-12 amino acid long linear peptides enriched in disordered regions of proteins that interact with domains of other proteins. The aim of this study was to search for novel targeting SLiMs in a dataset of proteins for which their localisation had already been experimentally determined.Check date.issued and date.embargo: Flexible delayed release embargo added by autho

Epigenetic enzymes influenced by oxidative stress and hypoxia mimetic in osteoblasts are differentially expressed in patients with osteoporosis and osteoarthritis

Epigenetic mechanisms including posttranslational histone modifications and DNA methylation are emerging as important determinants of bone homeostasis. With our case-control study we aimed to identify which chromatin-modifying enzymes could be involved in the pathology of postmenopausal osteoporosis and osteoarthritis while co-regulated by estrogens, oxidative stress and hypoxia. Gene expression of HAT1, KAT5, HDAC6, MBD1 and DNMT3A affected by oxidative stress and hypoxia in an in vitro qPCR screening step performed on an osteoblast cell line was analysed in trabecular bone tissue samples from 96 patients. Their expression was significantly reduced in patients with postmenopausal osteoporosis and osteoarthritis as compared to autopsy controls and significantly correlated with bone mineral density and several bone histomorphometry-derived parameters of bone quality and quantity as well as indicators of oxidative stress, RANK/RANKL/OPG system and angiogenesis. Furthermore, oxidative stress increased DNA methylation levels at the RANKL and OPG promoters while decreasing histone acetylation levels at these two genes. Our study is the first to show that higher expression of HAT1, HDAC6 and MBD1 is associated with superior quantity as well as quality of the bone tissue having a more favourable trabecular structure

Sex-determining region Y (SRY) attributes to gender differences in RANKL expression and incidence of osteoporosis

Receptor activator of nuclear factor kappaB ligand (RANKL) plays a crucial role in bone metabolism. RANKL gene misregulation has been implicated in several bone and cancer diseases. Here, we aimed to identify novel transcription regulators of RANKL expression. We discovered that transcription factors, sex-determining region Y (SRY) and c-Myb, regulate RANKL expression. We demonstrated that c-Myb increases and male-specific SRY decreases RANKL expression through direct binding to its 5\u27-proximal promoter. These results are corroborated by the gene expression in human bone samples. In osteoporotic men, expression of RANKL is 17-fold higher, which correlates with the drastically reduced expression (200-fold) of Sry, suggesting that in osteoporotic men, the upregulation of RANKL is caused by a decrease of Sry. In healthy men, the expression of RANKL is 20% higher than that in healthy women. Our data suggest that gender differences in RANKL expression and bone quality could be due to the sex-specific transcription factor SRY

Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma

Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 %s, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma

Gene Immunotherapy of Colon Carcinoma with IL-2 and IL-12 Using Gene Electrotransfer

Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin 12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory

Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome.

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation

Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation

Gene therapy in oncology, first steps of development in Slovenia

Genska terapija postaja čedalje bolj zanimiva tudi v onkologiji. Med aplikacijami je morda najzanimivejša imunostimulacija. Pripravimo lahko plazmidno DNA, ki nosi zapis za različne imunostimulatorne molekule, ki jih vnesemo v celice tumorjev ali normalnih tkiv. Ta tkiva postanejo proizvajalci teh molekul, ki lahko delujejo lokalno ali pa se izločajo tudi sistemsko v krvni obtok. Ker plazmidna DNA ne prehaja celične membrane, so potrebni dostavni sistemi, virusni ali nevirusni. V naših študijah uporabljamo predvsem nevirusni dostavni sistem – elektroporacijo. Interlevkin 12 (IL-12) je eden od zanimivih citokinov, za katerega je znano protitumorsko delovanje s spodbujanjem imunskega odziva in antiangiogenim delovanjem. Namen projekta SmartGene.si je bil pripraviti plazmid z zapisom za interlevkin 12 (plazmid phIL12) in pripraviti vse potrebno za njegovo klinično testiranje za zdravljenje kožnih tumorjev. V konzorciju smo združili moči s partnerji z akademskega in industrijskega področja. Treba je bilo pripraviti plazmid za uporabo v humani onkologiji po zahtevah Evropske agencije za zdravila (EMA). Za prijavo klinične študije na Javno agencijo za zdravila in medicinske pripomočke (JAZMP) smo morali izvesti tudi vse neklinične raziskave o varnosti in učinkovitosti zdravila. Nato je bilo treba razviti postopek priprave zdravila, zagotoviti primerne prostore za pripravo in izvedbo postopka priprave zdravila. V treh letih smo dosegli vse te zastavljene cilje in dobili dovoljenje za izvajanje klinične študije na kožnih tumorjih, ki ga je izdala JAZMP na osnovi pozitivnega mnenja Komisije Republike Slovenije za medicinsko etiko. Zdaj poteka klinična študija faze I preizkušanja plazmida phIL12 na kožnih tumorjih glave in vratu z namenom preveriti varnost in sprejemljivost genskega elektroprenosa plazmida v tumorje. Cilj študije je prav tako določiti primeren odmerek zdravila, ki bi ga v nadaljnji klinični študiji uporabili kot adjuvantno zdravljenje k ablativnim terapijam, kot sta radioterapija ali elektrokemoterapija.Gene therapy is also attracting interest in oncology. Probably the most interesting approach is immunostimulation. Plasmid DNA can be constructed, which is coding for a specific immunostimulatory molecule, which is then delivered into the cells, either in tumour or normal tissue. The transfected tissue then becomes the producer of the molecules encoded in the plasmid. The product is then released from the cells, either locally or systemically into the bloodstream. Since plasmids have hampered transport through the plasma membrane, delivery systems are needed that are either viral or nonviral. In our studies we predominantly use the non-viral transfection system, based on electroporation of the cells. Interleukin 12 (IL-12) is a cytokine with well-known anti-tumour and anti-angiogenic function. Therefore, in the SmartGene.si project we wanted to construct a plasmid DNA which is coding for IL-12 (plasmid phIL12), and perform all the necessary testing and prepare the documentation for its clinical testing in the treatment of skin tumours. The SmartGene.si consortium comprises partners from academia and industry. In the project it was necessary to prepare the plasmid according to the European Medicinal Agency (EMA) recommendations. For the application for the study approval submitted to the Agency for Medical Products and Medical Devices of the Republic of Slovenia (JAZMP), it was necessary to perform pharmacological, pharmacokinetic, and efficiency testing of phIL12. Thereafter, we had to develop the process and the facility, and prepare the drug. During the last three years, we have achieved all the goals and obtained the approval of the JAZMP for clinical testing of the product phIL12 in humans. We also obtained the approval of the National Ethics Committee. Currently, we are testing phIL-12 in a Phase I clinical protocol on head and neck skin tumours, with the aim to test the safety and feasibility of intratumoral gene electrotransfer of the plasmid phIL12. Another goal of the study is to determine a suitable dose of plasmid that could be used in future studies as adjuvant treatment to ablative therapies such as radiotherapy or electrochemotherapy