The brain and spinal cord make up the central nervous system (CNS), and as an immune-privileged site, it requires special immune surveillance and regulation. The complement system is a component of innate immunity produced locally in the CNS, since size restrictions from the blood brain barrier prevent complement proteins from easily passing through from the rest of the body. The complement pathway contributes to inflammatory cell recruitment, cell lysis, and opsonization, and thus requires regulation to avoid inappropriate activation. Despite its important role in innate immunity, very little is known about complement production, regulation, and function in the CNS of healthy or diseased individuals. For this dissertation, the central goal was to investigate and characterize the regulation of complement factor H (CFH), a regulator of the alternative pathway of complement activation. CFH polymorphisms have been associated with a number of diseases including atypical hemolytic syndrome, age-related macular degeneration, and Alzheimer's disease, but the regulation of CFH is not well understood, especially in the CNS. To investigate the role of CFH in the CNS, mRNA and protein production in glial cells was first established. The murine CFH (mCFH) promoter was cloned and the transcription start site was identified in astrocytes, microglia, and liver tissue. The mCFH promoter was truncated and different regions were investigated for enhancer or silencer activity. Database mining identified potential transcription factor binding sites, and mutagenesis studies and binding assays identified transcription factor binding candidates. Specifically, the activating protein-1 (AP-1) transcription factors c-Jun and c-Fos bound to a region of the mCFH promoter between – 416 base pairs (bp) and – 175 bp in an electrophoretic mobility supershift assay. Cytokine stimulation increased mCFH mRNA and protein production, as well as the mRNA production of c-Jun and c-Fos and the protein production of c-Jun. These results suggest a relationship between cell cycle and complement regulation, and the investigation of how these transcription factors and CFH affect disease will be a valuable area of research for CNS immune regulation.

Irrespective of plentiful efforts to enhance primary prevention and early detection, the number of melanoma cases in the United States has increased steadily over the past 30 years, thus greatly affecting public health and the economy. We have investigated the effects of five novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD), 3,4-Diaminonaphthalene-2,7-disulfonic acid (DNDA), [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocyte cell lines. Molecular docking data suggested that both ACPD and DNDA specifically bind to protein kinase C-zeta (PKC-ζ) and PKC-iota (PKC-ι) while both ICA-1 compounds specifically bind to PKC-ι, and ζ-Stat showed a high affinity towards PKC-ζ. Kinase activity assays were carried out to confirm these observations. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Both isoforms promote the activation of nuclear factor (NF)-κB and protein kinase B (AKT) thereby supporting survival and progression. In addition, we demonstrated that PKC-ι induced the metastasis of melanoma cells by activating Vimentin, and PKC-ι inhibition downregulated epithilial-mesencymal transition (EMT), while inducing apoptosis. Of note, PKC-ἱ specific inhibitors downregulated the expression of both PKC-ι and phosphorylated PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma. We also report the underlaying mechanisms of the transcriptional regulation of PKC-ι (PRKCI gene) expression in melanoma. c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC-ι regulation in SK-MEL-2 and MeWo cell lines. We silenced selected transcription factors using siRNA, and the results revealed that the silencing of c-Jun and FOXO1 significantly altered the expression of PRKCI. The levels of both phosphorylated and total PKC-ι increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun acts as an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. We also used a multiplex ELISA to analyze multiple pathways other than NF-κB that were affected by treatment with PKC-ι inhibitor. The silencing of NF-κB p65 and PKC-ι by siRNA suggested that the regulation of PKC-ι expression was strongly associated with FOXO1. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by these cytokines in an autocrine manner. Overall, the findings of this study suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling, while upregulating anti-tumor signaling, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma.

Stimulation of β-adrenergic receptors (βARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating βAR-stimulated apoptosis is not known. Stimulation of βARs with norepinephrine (10 μmol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6-fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 μmol/L) and Euk-134 decreased βAR-stimulated apoptosis by 89±6% and 76±10%, respectively. Infection with an adenovirus expressing catalase decreased βAR-stimulated apoptosis by 82±15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 μmol/L) decreased βAR-stimulated apoptosis by 76±8%, and the caspase inhibitor zVAD-fmk (25 μmol/L) decreased βAR-stimulated apoptosis by 62±11%. βAR-stimulated cytochrome c release was inhibited by MnTMPyP. βAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited βAR-stimulated apoptosis by 81±12%, and the JNK inhibitor SP600125 inhibited both βAR-stimulated apoptosis and cytochrome c release. Thus, βAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.

There is emerging evidence that the oncogenic potential of hdm2 (human and/or murine double minute-2 protein) stems not only from its ability to counteract tumor suppressor p53 but also from its less understood p53-independent functions. Surprisingly, little is known about the role and regulation of hdm2 in pancreatic tumors, a large proportion (50-75%) of which contain mutant p53. In this study, we determined that hdm2 was expressed in a Ras-signaling-dependent manner in various pancreatic cancer cell lines. As p53 was mutated and inactive in these cells, the expression of hdm2 was seemingly redundant. Indeed, the proliferation and survival of cell lines such as Panc-1 and Panc-28 could be inhibited by PRIMA-1 (mutant p53 activator) but not by Nutlin-3 (inhibitor of the hdm2-p53 interaction). Unexpectedly, however, the proliferation of both cell lines was strongly inhibited by hdm2-specific RNAi. Our data also revealed cyclin D1, c-Jun and c-Myc to be novel targets of hdm2 and suggested that they might mediate hdm2's role in cellular proliferation and/or survival. We conclude from our results that hdm2 is expressed in pancreatic cancer cells as a result of activated Ras signaling, and that it regulates cellular proliferation and the expression of three novel target genes by p53-independent mechanisms.

Stimulation of β-adrenergic receptors (β-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits β-AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with β1 integrins. Herein we tested the hypothesis that MMP-2 impairs β1 integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited β-AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated β-AR-stimulated decreases in mitochondrial membrane potential. Overexpression of β1 integrins using adenoviruses expressing the human β1A-integrin decreased β-AR-stimulated cytochrome c release and apoptosis. Overexpression of β1 integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the β1 integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis.

Breast cancer is the second most common malignancy in the world, accounting for over 1.7 million new diagnoses and an estimated 500,000 deaths per year (1). Overexpression of the receptor tyrosine kinase ErbB2, also known as Her2 or Neu, occurs in over 30% of breast cancers and correlates with metastasis, poor prognosis, and decreased survival (1, 2). Although therapeutics targeting ErbB2 show clinical efficacy, many patients display no initial response or develop drug resistance over time (2). A deeper understanding of the molecular basis of ErbB2-driven tumorigenesis is thus required for the development of improved therapeutic strategies. In vitro experiments suggest that activation of the c-Jun NH2-terminal kinase (JNK) pathway, a mitogen-activated protein kinase pathway, promotes proliferation, cellular invasion, and stem cell expansion in ErbB2-driven breast cancer (3, 4). Furthermore, unpublished data from our lab using mammary epithelial cells expressing activated ErbB2 show that JNK is required for acinus formation in in vitro 3D cultures. In contrast to these studies showing a tumorigenic role for the JNK pathway, other data from our lab show that JNK loss results in accelerated breast tumor growth, suggesting a tumor suppressive role (5, 6). However, these studies were performed in p53 knockout mice with or without a Kras mutation, where the latter required extensive aging and genomic instability to occur before differences in tumor growth were observable. To date, limited in vivo studies exist to confirm the role of JNK in more biologically relevant breast tumor models, such as in ErbB2-mediated cancer, which accounts for over 30% of all human breast cancers. In addition, the molecular mechanisms by which JNK signaling promotes ErbB2-driven tumorigenesis remains poorly understood. To address the discrepancy in JNK function between the in vitro ErbB2-driven breast cancer data and the in vivo p53 knockout tumor data, I began the development of an in vivo murine model to confirm the role of JNK in ErbB2-driven breast cancer. This mouse model will also allow us to test a potential mechanism by which JNK regulates tumorigenesis. Studies show that ErbB2-mediated secretion of the inflammatory cytokine IL6 promotes transformation and tumor growth by activation of the STAT3 transcription factor, triggering an IL6/STAT3 autocrine signaling loop (7,8). A major regulator of Il6 gene expression includes activator protein 1 (AP-1), a transcription factor composed of downstream JNK targets in the Jun protein family (9). In vitro experiments using ErbB2-overexpressing mammary epithelial cell lines show that chemical inhibition of JNK suppresses secreted IL6 protein levels, supporting a role for the JNK pathway in IL6 regulation (7). Thus, I hypothesize that JNK drives ErbB2-driven breast cancer by promoting IL6-mediated tumor progression. Addressing this will increase our understanding of the role of JNK in ErbB2-driven breast cancer and reveal a potentially new mechanism by which JNK functions in tumor progression. Additionally, I began the development of a mouse model that will allow us to investigate the role of JNK in macrophage polarization as an alternative mechanism by which JNK regulates ErbB2-driven breast cancer. In addition to promoting STAT3-dependent tumor growth, IL6 can indirectly drive tumorigenesis by promoting expression of the IL4 receptor in macrophages, triggering STAT6-mediated macrophage polarization towards the pro-tumorigenic M2 phenotype (10, 11). Unlike classically activated M1 macrophages, which promote inflammation and anti-tumor immunity, alternatively activated M2 macrophages function in immunosuppression and metastasis and correlate with advanced stages of breast cancer (12, 13). Further evidence supporting a role for the JNK pathway in macrophage polarization includes a recent study suggesting that JunB, a downstream JNK target and component of the AP-1 complex, plays a crucial role in the induction of M2 macrophage polarization in human alveolar macrophages (13). I hypothesize that activation of the JNK signaling pathway induces IL6-dependent macrophage polarization towards the pro-tumorigenic M2 phenotype. Addressing this hypothesis will determine for the first time whether JNK functions in regulating macrophage polarization within the tumor microenvironment, offering a potentially new mechanism by which JNK can promote ErbB2-driven breast cancer. Determining the role of JNK in ErbB2-mediated breast cancer will have direct therapeutic relevance, as targeting JNK has the potential to inhibit ErbB2-driven breast cancer and other IL6-mediated diseases. Investigating the underlying mechanisms by which JNK functions in ErbB2-positive breast cancer can also offer new molecular targets and further contribute to effective drug design.

Homocysteine (Hcy) has been identified as a primary risk factor for atherosclerosis as it induces endothelial cell (EC) activation/dysfunction and thus potentially initiating atherosclerotic plaque formation. There is accumulating evidence indicating a key role for oxidative stress in mediating Hcy atherogenic effects. The aim of this study was to evaluate the effects of chronic treatment with Hcy on EC activation and to explore the role of oxidative stress in these effects. Human umbilical vein endothelial cells (HUVEC) were cultured and treated chronically with DL-Hcy for 5-9 days. An in vitro flow system was also used to characterize the different types of interactions between DL-Hcy-treated HUVEC and neutrophils under physiological flow conditions. EC activation was studied by characterizing the activation of the JNK pathway and the up-regulation of different cell adhesion molecules (CAM) and cytokines, using different techniques including western blot, immunohistochemical staining, enzyme-linked immunosorbent assay and polymerase chain reaction. The role of oxidative stress was investigated by measuring the production of ROS and evaluating the efficiency of antioxidants. Furthermore, the role of nitric oxide and nitric oxide synthase in modulating Hcy effects was investigated. Chronic treatment with DL-Hcy did not kill the EC however, it inhibited cell proliferation. Furthermore, this treatment induced EC activation/dysfunction which was characterized by sustained activation of the JNK pathway, which in turn mediated up-regulation of E-selectin, ICAM-1 and to lesser extent P-selectin. Furthermore, DL-Hcy induced production of IL-8 protein. These CAM and chemokines collectively mediated different interactions between DL-Hcy-treated HUVEC and neutrophils under flow conditions including tethering, rolling, adherence and transmigration. DL-Hcy was also shown to induce significant ROS generation which mediated activation of the JNK pathway. Antioxidants restored DL-Hcy-induced interactions under flow to the basal level. DL-Hcy was shown to induce eNOS uncoupling which mediated, at least in part, the DL-Hcy-induced ROS production. Furthermore, short term treatment with NO inhibited DL-Hcy-induced HUVEC:neutrophil interactions in a cGMP-independent manner. In summary, this research showed that DL-Hcy has several proatherogenic effects, mediated at least in part by the JNK pathway, and induces EC activation/dysfunction priming for atherosclerosis initiation. The data supports that oxidative stress mediates the majority of Hcy atherosclerotic effects. Antioxidants tested, JNK inhibitors and NO showed promising results in reversing all DL-Hcy effects and restoring EC normal status. ¿

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Für die Progression des Wachstums maligner Tumoren und ihre Metastasierung ist die Angiogenese, die Bildung neuer Blutgefäße aus bereits existierenden, eine essentielle Voraussetzung. In dieser Arbeit konnte gezeigt werden, dass die proangiogenen Transkriptionsfaktoren c-Jun, Id1 und Id3 in ihrer Stabilität gegenüber dem Ubiquitin/26S-Proteasom-System durch das COP9-Signalosom (CSN) kontrolliert werden. Dieses bildet einen multimeren Proteinkomplex, der deutliche Homologien mit dem Lid-Subkomplex des 26S-Proteasoms aufweist. Sowohl c-Jun als auch Id3 binden an die Untereinheit CSN5. Id3 interagiert zusätzlich mit CSN7. Rekombinantes c-Jun, ein bekanntes Substrat der CSN-assoziierten Kinasen CK2 und PKD, wird durch Curcumin, einen Hemmstoff dieser Kinasen, deutlich destabilisiert. Daneben induziert Curcumin hochmolekulare Formen von c-Jun, bei denen es sich höchstwahrscheinlich um Ubiquitin-Konjugate handelt. Ferner beschleunigt Curcumin, ebenso wie die CK2- und PKD-Inhibitoren Emdodin, DRB und Resveratrol, in HeLa-Zellen den proteasomalen Abbau von c-Jun. Die c-Jun-abhängige Produktion von VEGF wird durch alle vier Kinase-Hemmstoffe signifikant reduziert. Verstärkt wird dieser Effekt noch durch den proteasomalen Inhibitor MG-132. Id3 wird nicht von den CSN-assoziierten Kinasen phosphoryliert. Allerdings hemmt es in einem Kinase-Assay die Phosphorylierung von c-Jun, ICSBP und CSN2. Curcumin und Emodin regen in HeLa-Zellen die Ubiquitinierung und den proteasomalen Abbau von Id3 an. Die Proteolyse von Id1 wird in HeLa-Zellen ebenfalls in Anwesenheit dieser beiden Hemmstoffe stimuliert. Mittels Kotransfektion von Id3 und His-markiertem Ubiquitin konnte eine verstärkte Ubiquitinierung von Id3 in Gegenwart von Curcumin direkt nachgewiesen werden. Außerdem wird Id3 durch die Überexpression von CSN2 stabilisiert. Auf diesen Daten basiert die Schlussfolgerung, dass die CSN-abhängige Phosphorylierung den Abbau von c-Jun und der beiden Id-Proteine über das Ubiquitin/26S-Proteasom-System inhibiert und dadurch ein interessantes neues Ziel einer antiangiogenen Tumortherapie repräsentiert. / Angiogenesis, the formation of new blood vessels from the existing vasculature, is a prerequisite for the progression of solid tumor growth and metastasis. In this study it is shown that the COP9 signalosome (CSN) regulates the stability of the angiogenic transcription factors c-Jun, Id1 and Id3 towards the ubiquitin/26S proteasome system. The COP9 signalosome constitutes a multimeric protein complex that shares sequence homology with the 26S proteasome lid complex. Both c-Jun and Id3 physically interact with the CSN subunit CSN5. In addition, Id3 can bind to CSN7. Recombinant c-Jun, a substrate of the CSN-associated kinases CK2 und PKD, is destabilized by curcumin, an inhibitor of these two kinases. Furthermore, curcumin induces high molecular weight c-Jun species, most likely ubiquitin conjugates. All tested inhibitors of the CK2 and PKD, emodin, DRB, resveratrol, as well as curcumin accelerate the degradation of c-Jun by the 26S proteasome in HeLa cells. The c-Jun-dependent expression of VEGF, the most potent angiogenic factor, is significantly reduced by the four kinase inhibitors. MG-132, an inhibitor of the 26S proteasome, also diminishes the production of VEGF. Id3 is not phosphorylated by the CSN-associated kinases. However, it inhibits c-Jun, ICSBP and CSN2 phosphorylation. Curcumin and emodin significantly induce ubiquitination and proteasome-dependent degradation of Id3 in HeLa cells. Proteasome-dependent degradation Id1 in HeLa cells is also stimulated by treatment with curcumin or emodin. Ubiquitination of Id3 is shown directly by cotransfection of HeLa cells with Id3 and His-tagged ubiquitin. Curcumin increases Id3-ubiquitin conjugate formation. In addition, overexpression of CSN2 leads to stabilization of Id3 protein. On the basis of these data it is concluded that CSN-mediated phosphorylation inhibits ubiquitination and proteasome-dependent degradation of c-Jun, Id1 and Id3. The COP9 signalosome thus represents an interesting new target for antiangiogenic tumor therapy.