This work focuses on anorexigenic neuropeptides, cocaine- and amphetamine-regulated transcript (CART) and prolactin-releasing peptide (PrRP), which decrease food intake and body weight. CART peptide is an anorexigenic neuropeptide and, despite many efforts, its receptor has not yet been identified. We found CART peptide specific binding sites in pheochromocytoma PC12 cells. Cells differentiated to neurons increased significantly the number of binding sites. On the other hand, after differentiation to chromaffin cells the number of binding sites was so low that it was impossible to determine their density. To clarify the importance of each of the three disulfide bridges in the CART molecule, analogs with one or two disulfide bridges were synthetized. The biological activity was maintained in analog with two disulfide bridges in positions 74-94 and 88-101. Moreover, we demonstrated the stimulation of JNK and subsequently c-Jun activation in PC12 cells. Neuropeptide PrRP belongs to the RF-amide peptide family and has anorexigenic properties. PrPR has a high affinity to GPR10 and neuropeptide FF (NPFF2) receptor. In our laboratory lipidized analogs of PrRP were synthesized, which are able to decrease food intake after peripheral administration and may cross the blood-brain barrier. We tested biological...

This work focuses on anorexigenic neuropeptides, cocaine- and amphetamine-regulated transcript (CART) and prolactin-releasing peptide (PrRP), which decrease food intake and body weight. CART peptide is an anorexigenic neuropeptide and, despite many efforts, its receptor has not yet been identified. We found CART peptide specific binding sites in pheochromocytoma PC12 cells. Cells differentiated to neurons increased significantly the number of binding sites. On the other hand, after differentiation to chromaffin cells the number of binding sites was so low that it was impossible to determine their density. To clarify the importance of each of the three disulfide bridges in the CART molecule, analogs with one or two disulfide bridges were synthetized. The biological activity was maintained in analog with two disulfide bridges in positions 74-94 and 88-101. Moreover, we demonstrated the stimulation of JNK and subsequently c-Jun activation in PC12 cells. Neuropeptide PrRP belongs to the RF-amide peptide family and has anorexigenic properties. PrPR has a high affinity to GPR10 and neuropeptide FF (NPFF2) receptor. In our laboratory lipidized analogs of PrRP were synthesized, which are able to decrease food intake after peripheral administration and may cross the blood-brain barrier. We tested biological...

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Type 1 diabetes is an autoimmune disease resulting in the selective destruction of the insulin producing β-cells in the pancreas. Pro-inflammatory cytokines and the free radical nitric oxide (NO) have been implicated in mediating the destruction of β-cells, possibly through activation of the mitogen activated protein kinases (MAPKs) JNK, ERK and p38. In addition to MAPKs, cytokine signaling also results in activation of the transcription factor nuclear factor-kappaB (NF-κB). The upstream signaling events leading to MAPK and NF-κB activation in β-cells are not well known. The work presented in this thesis therefore aims at characterizing the regulation of MAPKs and NF-κB in human islets, with emphasis on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. It was found that MEKK-1 was phosphorylated in response to the nitric oxide donor DETA/NONOate (DETA/NO), the β-cell toxin streptozotocin (STZ) and pro-inflammatory cytokines and that MEKK-1 downstream signaling in response to the same treatments involved activation of JNK but not ERK and p38. MEKK-1 was also found to be essential for cytokine-induced NF-κB activation. MEKK-1 downregulation protected human islet cells from DETA/NO-, STZ, and cytokine-induced cell death. Furthermore, overexpression of the NF-κB subunit c-Rel protected human islet cells from STZ and hydrogen peroxide-induced cell death indicating that NF-κB activity protects against cell death in human islets. In summary, these results support an essential role for MEKK-1 in the activation of JNK and NF-κB, with important consequences for human islet cell death and that strategies preventing human islets death by inhibition of the JNK pathway instead of NF-κB might be suitable.

Η θεραπεία της Οξείας Μυελογενούς Λευχαιμίας (ΟΜΛ) είναι συχνά ανεπιτυχής λόγω ανάπτυξης κυτταρικής αντίστασης στα αντιλευχαιμικά φάρμακα. Εκτός από την έκφραση Ρ-γλυκοπρωτείνης στα λευχαιμικά κύτταρα, άλλοι κυτταρικοί παράγοντες μπορούν επίσης να συμβάλλουν στην χημειοαντίσταση. Η c- Jun N-terminal Kinase (JNK) είναι μία πρωτεινική κινάση που ενεργοποιείται όταν τα κύτταρα εκτεθούν σε χημειοθεραπευτικά φάρμακα (ΧΜΘ). Πρόσφατες μελέτες σε συμπαγείς όγκους συσχετίζουν την χημειοαντίσταση με αδυναμία των καρκινικών κυττάρων να ενεργοποιήσουν τη JNK κατόπιν επίδρασης ΧΜΘ. Σκοπός της εργασίας είναι να διερευνήσει αν η χημειοαντίσταση στην ΟΜΛ οφείλεται σε ενδογενή αδυναμία των λευχαιμικών βλαστών να ενεργοποιήσουν τη JNK. Μεθοδολογία: Συγκρίναμε ευαίσθητες (U937) και ανθεκτικές (U937R) στις ανθρακυκλίνες κυτταρικές σειρές ΟΜΛ ως προς την δυνατότητα in vitro ενεργοποίησης της JNK κατόπιν επίδρασης ΧΜΘ (Western Blot). Επιπλέον, στις λευχαιμικές κυτταρικές σειρές ελέγξαμε απευθείας τη σημασία της JNK στην χημειοαντίσταση με πειράματα α) αποσιώπησης της JNK με JNK1–στοχεύον siRNA και β) ενεργοποίησης της JNK (διαμόλυνση με τον ΜΚΚ4/SEK1 άνωθεν ενεργοποιητή της JNK) Περαιτέρω, ελέγξαμε την in vitro δυνατότητα ενεργοποίησης της JNK σε 29 πρωτογενή μυελικά δείγματα ΟΜΛ κατόπιν βραχείας διάρκειας (30-60min) έκθεση στην daunorubicin (1μΜ) και συσχετίσαμε τα εργαστηριακά δεδομένα με κλινικά χαρακτηριστικά των ασθενών με ΟΜΛ. Αποτελέσματα: In vitro θεραπεία των U937 κυττάρων με ανθρακυκλίνες προκάλεσε ισχυρή και ταχεία ενεργοποίηση της JNK και απόπτωση. Αντίθετα, στα πολυανθεκτικά U937R κύτταρα δεν παρατηρήθηκε ενεργοποίηση της JNK, ακόμη και σε συνθήκες υψηλής ενδοκυττάριας συγκέντρωσης ανθρακυκλινών. Αποσιώπηση της JNK στα ευαίσθητα U937 κύτταρα τα έκανε ανθεκτικά στις ανθρακυκλίνες (JNK1-siRNA διαμολυσμένα U937 κύτταρα εμφάνισαν 50.4% και 61.3% ελαττωμένη daunorubicin- (DNR, 1μΜ 24hr) και doxorubicin- (DOX, 1.5μΜ 24hr) προκαλούμενη απόπτωση αντίστοιχα, συγκριτικά με U937 κύτταρα-μάρτυρες, P<0.001). Αντίστροφα, εκλεκτική ενεργοποίηση της ανενεργού JNK στα ανθεκτικά U937R κύτταρα τα έκανε 3.3 φορές πιο ευαίσθητα στη DNR και 3.1 φορά πιο ευαίσθητα στη DΟΧ, συγκριτικά με U937R κύτταρα-μάρτυρες. Επιπρόσθετα, παρατηρήσαμε ισχυρή συσχέτιση μεταξύ των in vitro φαρμακοδυναμικών αλλαγών των επιπέδων ενεργοποίησης της JNK στους λευχαιμικούς βλάστες και της ανταπόκρισης των ασθενών με ΟΜΛ στη χημειοθεραπευτική αγωγή (P=0.012). Η απουσία ενεργοποίησης της JNK στα βλαστικά κύτταρα συσχετίστηκε επίσης με αρνητικούς προγνωστικούς παράγοντες για την ΟΜΛ, όπως γηραιότερη ηλικία των ασθενών (P=0.046) και ΟΜΛ αναπτυσσόμενη επί εδάφους μυελοδυσπλασίας (P=0.017). Συνοψίζοντας, τα in vitro και in vivo αποτελέσματα μας προτείνουν την ενδογενή αποτυχία ενεργοποίησης της πρωτεινικής κινάσης JNK στους λευχαιμικούς βλάστες σαν έναν εναλλακτικό μηχανισμό χημειοαντίστασης στην ΟΜΛ. Η διελεύκανση των μηχανισμών εκείνων που επιφέρουν καταστολή της JNK στην χημειοανθεκτική ΟΜΛ μπορεί να ωφελήσει θεραπευτικά. / Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug-resistance in AML. c- Jun N-terminal Kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

[Truncated abstract] Reactive oxygen species (ROS) have been implicated to play a major role in many pathological conditions including heart attack and stroke. Their ability to modulate the extracellular signal-regulated protein kinase (ERK) and c-Jun Nterminal kinase (JNK) signalling pathways, thereby influencing cellular response has been well-documented. Recent studies implicate a central role for mitochondria in ERK and JNK activation by ROS although the mechanisms remained unresolved. Using Jurkat T-lymphocyte as a cell model, this study demonstrated increased mitochondrial ROS production as a result of decreased mitochondrial complex activities mediated by hydrogen peroxide treatment. This is the first study to show that mitochondria are not essential for activating ERKs, however damaged mitochondria producing ROS can be expected to cause sustained ERK activation . . . This study revealed that JNK and its upstream kinases MKK4, MKK7 and ASK1 are associated with the mitochondria. Furthermore, findings from this study imply that JNK resides in the mitochondrial matrix. This study is the first to demonstrate that mitochondrial JNK can be activated in a cell-free environment by signals originating from the mitochondria. Experimental work using isolated mitochondria demonstrated that mitochondrial JNK can be activated by ROS generated from the mitochondria themselves. Flavin-containing proteins appear to be the main sources of mitochondrial-ROS which signal through redoxsensitive proteins to activate mitochondrial JNK.

Thesis (Ph.D. in Immunology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 190-212). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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The JNK signal transduction pathway mediates a broad, complex biological process in response to inflammatory cytokines and environmental stress. These responses include cell survival and apoptosis, proliferation, tumorigenesis and the immune response. The divergent cellular responses caused by the JNK signal transduction pathway are often regulated by spatial and cell type contexts, as well as the interaction with other cellular processes. The discovery of additional components of the JNK signal transduction pathway are critical to elucidate the stress response mechanisms in cells. This thesis first discusses the cloning and characterization of two novel members of the JNK signal transduction pathway. JIP1 and JMP1 were initially identified from a murine embryo library through a yeast Two-Hybrid screen to identify novel JNK interacting proteins. Full length cDNAs of both genes were cloned and analyzed. JIP1 represents the first member of the JIP group of JNK scaffold proteins which were characterized. The JNK binding domain (JBD) of JIP1 matches the D-domain consensus of other JNK binding proteins, and it demonstrates JNK binding both in vitro and in vivo. This JNK binding was demonstrated to inhibit JNK signal transduction and over-expression of JIP1 inhibits the JNK mediated pre-B cell transformation by bcr-abl. Over-expressed JIP1 also sequesters JNK in the cytoplasm, which may be a mechanism of the inhibition of JNK signaling. A new, high-resolution digital imaging microscopy technique using deconvolution demonstrated the absence of JNK1 in the nucleus of co-transfected JIP1 and JNK1 cells. The other protein discussed in this thesis is JMP1, a novel JNK binding, microtubule co-localized protein. There is a JBD in the JMP1 carboxyl end and a consensus D-domain within this region. The JMP1 JBD demonstrates an increased association with phospho-JNK from UV irradiated cells compared to un-irradiated cells in vivo. JMP1 also has 12 WD-repeat motifs in its amino terminal end which are required for microtubule co-localization. JMP1 demonstrates a cell cycle specific localization at the mitotic spindle poles. This co-localization is dependent on intact microtubules and the amino-terminal WD-repeats are required for this localization. JMP1 mRNA is highly expressed in testis tissues. Immunocytochemistry on murine testis sections using an affinity purified anti-JMP1 antibody demonstrates JMP1 protein in the lumenal compartment of the seminiferous tubules. JMP1 protein is expressed in primary and secondary spermatocytes, cells which are actively undergoing meiosis. The results obtained from the localization of JMP1 in meiotic spermatocytes led to an investigation of the roles of JNK signal transduction in the testis. The testis is an active region of cellular proliferation, apoptosis and differentiation, which make it an appealing model for studying JNK signal transduction. However, the roles JNK signaling have in the testis are poorly understood. I investigated the reproduction capability of Jnk3-/- male mice and discovered older Jnk3-/- males had a reduced capacity to impregnate females compared to younger animals and age-matched wild type controls. The testis morphology and sperm motility of these animals were similar to wild-type animals, and there was no alteration of apoptosis in the testis. The final section of this thesis involves the study of this breeding defect and investigating for cellular defects that might account for this age-related Jnk3-/- phenotype.

Abstract Wnt genes encode secreted signalling molecules that control embryonic development including organogenesis, while dysregulated Wnt signalling is connected to many diseases such as cancer. Specifically, Wnts control a number of cellular processes such as proliferation, adhesion, differentiation and aging. Many Wnt proteins activate the canonical β-catenin signalling pathway that regulates transcription of a still poorly characterized set of target genes. Wnts also transduce their signaling in cells via β-catenin-independent “non-canonical” pathways, which are not well understood. In this study, Wnt-11 signalling mechanisms in a mammalian model cell line and roles of Wnt-11 in heart development were analyzed in detail. In addition the aim was to identify new Wnt target genes by direct chromatin immunoprecipitation and Affymetrix GeneChip assays in the model cells exposed to Wnt-3a. Our studies reveal that Wnt-11 signalling coordinates the activity of key cell signalling pathways, namely the canonical Wnt/β-catenin, the JNK/AP-1, the NF-κB and PI3K/Akt pathways in the CHO cells. Analysis of the Wnt-11-deficient embryos revealed a crucial role in heart organogenesis. Wnt-11 signalling coordinates cell interactions during assembly of the myocardial wall and Wnt-11 localizes the expression of N-cadherin and β-catenin to specific cellular domains in the embryonic ventricular cardiomyocytes. Collectively these studies reveal that the mammalian Wnt-11 behaves as a non-canonical Wnt and that it is a critical factor in the coordination of heart development. Specifically, it controls components of the cell adhesion machinery. Analysis of the Wnt target genes revealed a highly context-dependent profile in the Wnt-regulated genes. Several new putative target genes were discovered. Out of the candidate Wnt target genes, Disabled-2 was identified as a potential new direct target for Wnt signalling.