doktorska disertacija
Teja Čelhar Kenanova (Author), Irena Mlinarič-Raščan (Mentor)

Abstract

Nepravilnosti v odzivu limfocitov B na dražljaje tekom zorenja ali aktivacije vodijo do avtoimunskih obolenj, limfomov in levkemij. B-celični odziv je natančno reguliran proces, v katerem imajo ključno vlogo premreženje receptorja BCR, T-celično posredovani signali, stimulacija receptorjev TLR ter prisotnost citokinov in drugih ligandov za membranske receptorje, kot so na primer prostaglandini, v mikrookolju limfocitov B. Identifikacija novih molekul, ki sodelujejo v kompleksni signalni mreži limfocitov B, bo pripomogla k boljšemu razumevanju B-celičnega odziva in izboru obetavnih tarč za razvoj ciljanih terapij. V okviru doktorskega dela smo kot zanimive tarče za modulacijo apoptoze in proliferacije celic limfoma B predstavili serinske proteaze ter prostaglandinski receptor EP4. V prvem delu smo testirali nabor inhibitorjev serinskih proteaz, sintetiziranih na Fakulteti za farmacijo, na zmožnost indukcije apoptoze na človeških in mišjih linijah limfoma B. Ugotovili smo, da so spojine, zasnovane na azafenilalaninskem skeletu, citotoksične za celice limfoma B in povzročajo apoptotično celično smrt. Te spojine so povzročile biokemijske in morfološke značilnosti apoptoze: aktivacijo kaspaz, izgubo mitohondrijskega potenciala, razpad celičnega jedra in fragmentacijo DNA. Večina inhibitorjev je bila selektivnih za trombin, s konstantami inhibicije v nanomolarnem območju, vendar so močno inhibirali vsaj še eno dodatno serinsko proteazo (tripsin, kimotripsin in/ali faktor koagulacije X). V drugem delu doktorskega dela smo se osredotočili na prostaglandinski receptor EP4, ki v limfocitih B posreduje inhibitorne učinke endogenega liganda PGE2. Iz naših rezultatov lahko sklepamo, da agonist receptorja EP4 modulira učinke posredovane preko receptorjev BCR in TLR4. Stimulacija receptorjev BCR in TLR4 namreč poveča ekspresijo EP4, stimulacija receptorja EP4 pa nato kot pozitivna povratna zanka pospešuje učinke specifične vezave antigena na receptor BCR in kot negativna povratna zanka zavira nespecifično aktivacijo preko poliklonalnega aktivatorja LPS. Vsaj del teh učinkov je posredovan preko inhibicije aktivacije transkripcijskega faktorja NFκB. Nizke koncentracije ireverzibilnega inhibitorja serinskih proteaz TPCK prav tako zavirajo aktivacijo NFκB, ne da bi signifikantno vplivale na proliferacijo celic WEHI-231.Kombinacija razmeroma netoksičnih koncentracij TPCK in agonista EP4 (PGE1-OH) pa je za celice usodna, saj povzroči popolno smrt celic. Predvidevamo, da je za ta učinek ključna sinergistična inhibicija aktivacije NFκB.

Keywords

homeostaza limfocitov B;apoptoza;receptor EP4;inhibitorji serinskih proteaz;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FFA - Faculty of Pharmacy
Publisher: [T. Čelhar Kenanova]
UDC: 616-074
COBISS: 2692977 Link will open in a new window
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Other data

Secondary language: English
Secondary title: ǂThe ǂrole of serine proteases and prostaglandin receptor EP4 in the modulation of apoptosis in B lymphocytes
Secondary abstract: Many diseases of the immune system such as autoimmune diseases, lymphomas and leukemias are caused by defects in the process of B cell maturation and activation. Various signals contribute to the tightly regulated B cell response, among which are crosslinking of BCR, T-cell help, TLR stimulation and the presence of cytokines and other ligands for membrane receptors, such as prostaglandins, in the B cell microenvironment. The identification of novel molecules that participate in the complex B cell signaling network will contribute to a better understanding of B cell responses and give rise to novel molecular targets for drug discovery and development. In the present work we present two novel molecular targets for the modulation of B cell responses: serine proteases and the prostaglandin receptor EP4. In the first part of our research we tested if a series of inhibitors of serine proteases synthesized at the Faculty of Pharmacy were able to induce apoptotic cell death in human and murine B lymphoma cell lines. We have shown that compounds built on an azaphenylalanine scaffold are severely cytotoxic for B-cell lymphoma cells and they provoke biochemical and morphological changes typical of apoptosis, such as DEVDase activation, loss of mitochondrial membrane potential, nuclear degradation and genomic DNA fragmentation. Most of the inhibitors proved to be selective for thrombin, with inhibition constants (Ki) in the nanomolar range. However, they could also inhibit at least one additional serine protease (trypsin, chymotrypsin and/or coagulation factor X) with Ki values in the nanomolar or low micromolar range. These serine protease inhibitors constitute novel apoptosis inducing compounds in B-cell lymphoma cells. In the second part of our work we focused on the prostaglandin receptor EP4, which is the principal molecule conveying the growth-suppressive effect of prostaglandin E2. Our results suggest that an agonist of the EP4 receptor modulates the effects mediated by the receptors BCR and TLR4. We have shown that both receptors up-regulate the expression of EP4, which stimulation can in turn act as a positive feedback loop increasing the inhibitory effect of BCR crosslinking and as a negative feedback loop suppressing the proliferatory effect of TLR4 signaling. In WEHI-231 cells we demonstrated that this effect is at least in part mediated by the inhibition of activation of the transcription factor NFκB. Non-cytotoxic concentrations of the irreversible inhibitor of serine proteases TPCK were also able to inhibit NFκB activation. However, the combination of low concentrations of TPCK and an EP4 agonist (PGE1-OH) proved to be lethal for WEHI-231 cells, causing cell death. We suggest that this effect is caused by a synergistic inhibition of NFκB activation.
Type (COBISS): Dissertation
Thesis comment: Univ. Ljubljana, Fak. za farmacijo
Pages: XI, 142 str.
ID: 12895816