The NF-B transcription factor family plays a central role in many aspects of the immune response, and activation of this family of transcription factors has been shown to trigger many disease processes. Thus, the ability to modulate NF-kB activity may be an attractive way to treat these diseases. We used an in vitro cell-based assay to test potential NF-kB inhibitors by measuring their effect on IL-1-induced expression of the NF-B dependent intracellular adhesion molecule-1 (ICAM-1, CD54). To develop the cell-based system we sorted IL-1b-responsive U373 human astrocytoma cells to obtain a population of cells with minimum background expression and maximum induced expression of CD54 following stimulation with IL-1. We tested ethyl pyruvate, a novel anti-inflammatory drug candidate, and the ability of related compounds to block activation of NF-kB activity by measuring the expression of CD54 on U373 cells exposed to IL-1. 4-hydroxyphenylpyruvic acid was the best inhibitor of CD54 upregulation. We further tested the compounds using the mouse macrophage-like RAW 264.7 cell line which produce a variety of cytokines and nitric oxide (NO) following exposure to lipopolysaccharide (LPS) in an NF-kB-dependent manner. The drugs downregulated LPS-induced IL-6 production, iNOS upregulation, and NO production following the same efficacy trend observed in the primary screening using CD54 expression in U373 cells. These studies show the ease of using an endogenous reporter gene (i.e., CD54) and FACS analysis to rapidly characterize the relative efficacy of pharmacologic inhibitors. A second completely unrelated topic of the dissertation dealt with the receptor for advanced glycation end-products (RAGE). RAGE is thought to be important in a variety of pathological conditions, including diabetes, sepsis, atherosclerosis, renal diseases, hypertension and Alzheimers disease. However, RAGE proximal signaling events are still unclear. We were able to establish that original RAGE, sequenced from bovine lung, is only present in the lung. This observation was based on antibody specificity, Northern blotting and N-glycosylation analysis. One of the antibodies that we used (H-300, Santa Cruz, CA) was very selective for lung RAGE and not cross-react with other RAGE isoforms. Only lung RAGE had a transcript size of 1.4 kb as determined by Northern blot and only lung RAGE was N-glycosylated. Non-lung tissues and cell lines appeared to express their own unique RAGE isoforms. Non-lung derived cell lines were permissive for lung RAGE isoform expression but lung derived cell lines were not. Interestingly, all transfected cell lines (of lung and non-lung origin) expressed RAGE mRNA transcripts. In addition, we established that previously described endogenous soluble RAGE (esRAGE) does not contain any of the canonical RAGE epitopes, but includes sequence encoded in intron 9. RAGE knockout mice lose esRAGE isoform along with the canonical one confirming that esRAGE originates from the RAGE gene. Signaling studies with pro-inflammatory stimuli in mouse lung slices of wild-type and knockout mice revealed the importance of RAGE in LPS and IL-1-induced inflammatory response, but not when reported RAGE ligands, including AGEs, HMGB1 and S100B, were applied.

Background Prostate cancer is the second leading cause of cancer related death in men. Current therapies for metastatic prostate cancer can only prolong progression, as most men eventually succumb to metastasis and then death. Therefore, there is continued urgency to identify novel therapeutic targets for advanced disease. Previous reports have identified an increase in Translocator Protein (TSPO) expression in numerous cancer models, including prostate. Functionally, TSPO has been implicated in the regulation of apoptosis and cell proliferation. Here, the role of TSPO in advanced prostate cancer is evaluated in an effort to establish the potential value of TSPO as a therapeutic target in advanced disease. Methodology and Principle Findings Immunohistochemical analysis using tissue microarrays was used to determine the expression profile of TSPO in human prostate cancer tissues. We observed that TSPO expression increases with disease progression, as prostate cancer metastases had the highest expression. To demonstrate the effect of TSPO ligands PK11195 and lorazepam in prostate cancer, we utilized cell proliferation assays, cell death ELISAs, and a prostate cancer mouse xenograft study. Our findings provide the first evidence of the anti-tumor effects of lorazepam acting on TSPO. To determine the effect of modulating TSPO expression, we performed overexpression and knockdown studies. These studies provided further evidence that lorazepam is acting through TSPO, as overexpression of TSPO conferred increased susceptibility to lorazepam while TSPO knockdown decreased susceptibility. Lastly, we investigated the role of TSPO multimers in prostate cancer. We found that TSPO multimers can be induced by reactive oxygen species and may be formed through a di-tyrosine covalent bond. Conclusions and Significance TSPO expression increases with prostate cancer progression. The benzodiazepine lorazepam exerts its anti-cancer effects through its binding to TSPO. Collectively, these data suggest that TSPO is an excellent therapeutic target for advanced disease and that our preclinical results demonstrating that the already existing FDA-approved drug lorazepam has anti-tumor effects could be easily translated to the prostate cancer patient population. These studies could lead to a significant change in the management of prostate cancer by providing a treatment option with minimal toxicity for use in advanced disease and could ultimately prevent prostate cancer deaths.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super-family of cell surface receptors whose activation has been suggested to contribute to various pathologies. RAGE has been primarily studied in diabetes where its upregulation has been linked to disease in the kidney, vasculature, and nervous system. This protein is highly expressed in the lung under normal conditions, but its function is unknown. We therefore investigated the normal function of RAGE in the lung and its pulmonary expression in two disease states. Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with both high morbidity and mortality. Unfortunately, there are currently no effective therapies for IPF necessitating mechanistic insight into the disease pathogenesis. We found that pulmonary fibrosis led to a depletion of RAGE in both animal models and tissue from patients with idiopathic pulmonary fibrosis. In contrast to other diseases in which RAGE signaling promotes pathology, we found that aged RAGE null mice spontaneously develop pulmonary fibrosis-like alterations and more severe fibrosis in response to asbestos injury. In addition, we found that RAGE null mice were fully protected from the fibrotic effects of bleomycin. In addition, we investigated the expression of RAGE in the lungs of diabetic rodents. Diabetes has been shown to alter RAGE expression in a number of tissues that do not normally express RAGE. We hypothesized that diabetes would alter pulmonary RAGE expression and contribute to the susceptibility to pulmonary injury. We found that pulmonary RAGE expression was unaltered in five rodent models suggesting that diabetes does not effect RAGE expression in the lung. Lastly, we identified that RAGE has a very high affinity for components in the basement membrane of the lung. A few RAGE studies suggested that it might serve a role as an adhesion molecule. We found that RAGE extensively colocalized with the alveolar basement membrane and had very high affinity for collagen I, collagen IV, and laminin, but not fibronectin. These findings along with the fact that RAGE null mice spontaneously develop fibrosis suggest a potential homeostatic function of RAGE in the lung. This is in stark contrast to the vast majority of studies, which suggest that its expression is solely pathologic.

Integrin-mediated cell-extracellular matrix (ECM) adhesion is essential for the survival of normal epithelial cells, and loss of this cell-ECM adhesion leads to anoikis. In this dissertation study, we first identify migfilin, a novel focal adhesion protein, as a key sensor of cell-ECM adhesion in epithelial cells. Loss of cell-ECM adhesion significantly reduces migfilin protein levels in untransformed epithelial cells and concomitantly induces anoikis. Migfilin RNAi is sufficient to induce apoptosis in MCF-10A cells while overexpression of FLAG-migfilin partially protects these cells from anoikis, strongly suggesting that migfilin plays a critical role in cell adhesion-mediated cell survival signaling. Cell detachment-induced migfilin reduction is, at least partially, responsible for the induction of anoikis. Further signaling studies reveal that migfilin regulates cell survival and anoikis by influencing Src activation. Immunoflorescence staining shows that migfilin co-localizes with active Src in focal adhesions, and immunoprecipitation and GST pull-down assays demonstrate that migfilin directly interacts with Src. Moreover, the detailed structural studies show that migfilin strongly binds to the Src SH3 domain via the second PXXP cluster (140-173aa) in its proline-rich region, and weakly binds to the Src SH2 domain via an atypical binding sequence (E6KRVASS12) in its N-terminal. A working model is proposed in which migfilin promotes Src activation via direct interaction, and loss of cell-ECM adhesion triggers the degradation of migfilin protein, thereby causing Src inactivation which contributes to the initiation of anoikis. Interestingly, this migfilin-Src signaling pathway is dysfunctional in some anoikis-resistant cancer cells. During cell detachment, migfilin proteins are stabilized in these cancer cells, and phosph-Y419 Src levels are not reduced concomitantly, representing a novel mechanism for anoikis resistance during tumorigenesis. In addition, migfilin is found to negatively regulate p27 protein level. Depletion of migfilin significantly increases p27 protein levels in different cell lines. In HCT116 cells, migfilin RNAi increases both cytoplasmic and nuclear p27, and inhibits cell cycle progression. These findings indicate that migfilin provides a linkage between p27 and integrin-mediated cell-ECM adhesion.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide due to poor prognosis and limited therapeutic options. Point mutations affecting phosphorylation sites such as serine-45 in Beta-catenin gene are evident in around 30% of HCC. We developed a transgenic mouse that expresses Ser45 mutated Beta-catenin (TG) in hepatocytes. While this mutation did not induce spontaneous tumorigenesis, it promoted diethylnitrosamine (DEN)-induced HCC through cyclin-D1 overexpression and other factors. The Wnt/Beta-catenin signaling is important in stem cell self-renewal. The adult progenitor cell of the liver, or oval cells that emanate from atypical ductular proliferation (ADP), maybe involved in liver regeneration and/or hepatocarcinogenesis and can be observed after exposure to DDC diet, which induces hepatic and biliary injury. When challenged with chronic-DDC diet, Beta-catenin transgene led to a cellular disparity in the form of increased appearance of atypical hepatocytes (positive for ductular marker A6), which was associated with better resolution of intrahepatic cholestasis. We also utilized DDC diet in conditional Beta-catenin knockout mice (KO) that lacked Beta-catenin in hepatocytes and cholangiocytes. ADP was blunted after short-term DDC feeding in KO mice; however, long-term feeding resulted in gradual increase in ADP, hepatic fibrosis and HCC. Interestingly, the KO livers begin to exhibit periportal Beta-catenin-positive hepatocytes, which eventually populate the entire livers over the course of this process. Finally, we explored targeting of the Wnt pathway with pegylated interferon-alpha2A (Peg-IFN). We found that Peg-IFN decreased Beta-catenin activity in mouse liver and several human hepatoma cell lines. The mechanism seemed to be at least partly due to upregulation of a nuclear export factor, RanBP3. Thus, this study characterizes an animal model utilizing Beta-catenin mutation, which is evident in HCC patients. DEN-exposure in these animals led to HCC development, thus providing a valuable tool to study mechanisms of hepatocarcinogenesis and providing a model to test therapeutic inhibition of Beta-catenin by agents such as peg-IFN and others. Our studies also provide evidence that Wnt activation may resolve intrahepatic cholestasis. Finally, we show that chronic damage to the liver in KO led to appearance of Beta-catenin-positive hepatocytes, which continued to proliferate and in the face of continued injury and fibrosis, led to development of HCC, which is also relevant clinically.

Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol-anchored, cell-surface heparan sulfate proteoglycans. The GPC3 gene is located on the X chromosome, and is highly expressed during embryogenesis and organogenesis. Loss-of-function mutations of GPC3 in humans result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and post-natal liver and other organ overgrowth. GPC3 is one of the most over-expressed proteins in human hepatocellular carcinoma and is used as a novel diagnostic marker. However, its role in normal liver regeneration is still not well characterized. In this study, we investigated the role and effects of GPC3 in hepatocyte proliferation and liver regeneration, using 2/3 partial hepatectomy (PHx) model in rats and hepatocyte-targeted GPC3 transgenic mice. We found in rats that GPC3 mRNA and protein increase in a time frame which coincides with the termination of proliferative activities of either hepatocytes (day 2 after PHx and day 8-12 in culture) or non-parenchymal cells (day 5-6 after PHx). Blocking GPC3 expression using morpholino oligonucleotides promoted rat hepatocyte growth in vitro. We further generated GPC3 transgenic mice with hepatocyte-targeted over-expression of GPC3. These transgenic mice develop normally compared with their non-transgenic littermates, but have a suppressed rate of hepatocyte proliferation and liver regeneration after 2/3 PHx. Therefore we hypothesize that GPC3 is a negative regulator of hepatocyte proliferation and liver regeneration. The yeast two-hybrid assay revealed that GPC3 interacts with several interesting proteins including CD81, a cell membrane tetraspanin. CD81 levels changed in the same manner as GPC3 after rat PHx, and their interaction was confirmed by co-immunoprecipitation and co-immunofluorescence. The co-localization of GPC3 and CD81 after PHx indicates an important regulator interaction between the two proteins. Moreover, gene array analysis revealed a series of changes in the expression profiles in GPC3 transgenic mice. After PHx, a panel of cell cycle related genes and some oncogenes are either up- or down-regulated, which was confirmed by western blotting. Our results indicate that GPC3 plays a negative regulatory role in hepatocyte proliferation and liver regeneration in rats and hepatocyte-targeted transgenic mice, in which several potential proteins and multiple pathways are involved and affected.

Amyotrophic lateral sclerosis (ALS) is the most common form of adult onset motor neuron disease and is characterized by the progressive degeneration and death of motor neurons. The pathologic mechanisms underlying ALS are poorly understood although our laboratory identified decreased levels of transthyretin (TTR), a protein that impacts the retinoid signaling pathway, in the cerebrospinal fluid of ALS patients. Differential expression of retinoid signaling components has been reported in ALS patients and transgenic animal models of familial ALS. We sought to further characterize TTR and retinoid signaling proteins in ALS and to evaluate the role of retinoid signaling in motor neuron cell death. Mass spectrometry and immunoblotting were used to investigate TTR. Immunohistochemistry using lumbar spinal cord tissue from ALS patients and non-neurologic disease controls was used to characterize retinoid signaling pathway proteins. Spinal cord tissue homogenates were used for co-immunoprecipitation studies and electrophoretic mobility shift assays. Motor neuron-enriched cultures established from embryonic day 14 rats were utilized for in vitro studies. RAR-mediated signaling was modulated with pan-agonists and isotype-specific agents and hydrogen peroxide used to model oxidative stress/injury. Altered post-translational modifications and high molecular weight species of the TTR protein were observed in ALS. Cellular retinoic acid binding protein-II (CRABP-II) and retinoic acid receptor beta (RARβ) exhibited increased nuclear localization in motor neurons of sporadic ALS patients. Protein-protein interactions (between CRABP-II and RARα or RARβ) did not differ although retinoic acid response element binding was increased in ALS as compared to controls. Treatment with a pan-RAR or RARβ-specific agonist significantly decreased oxidative stress-induced motor neuron cell death in vitro and genes downstream of RARβ were increased with treatment. Our results indicate that TTR genetic polymorphisms do not represent a novel susceptibility factor for ALS, although protein modification and aggregation appear to be altered in ALS. Localization of proteins of the retinoid signaling pathway is altered in ALS patients and these changes translate to the transcriptional level. Our in vitro work indicates that stimulating the RARs (particularly RARβ) is neuroprotective and that pharmacologic agents that target this nuclear receptor may be of value in slowing the progression of ALS.

Beta-catenin plays multiple roles in liver health and disease through regulation of proliferation, differentiation and metabolism. Elucidating the molecular basis of how beta-catenin regulates these diverse functions and others is the subject of this dissertation. While beta-catenin signaling undergoes temporal activation and its loss dampens liver regeneration (LR), the impact of stimulating this pathway remains unknown. We utilized transgenic (TG) mice expressing Ser45 mutated beta-catenin in hepatocytes to show a growth advantage both in vitro and during LR through cyclin-D1 regulation. Additionally, hydrodynamic delivery of Wnt-1 gene delivery induced beta-catenin activation and hepatocyte proliferation during LR. Regucalcin or senescence marker protein-30 (SMP30) was identified as a beta-catenin target in the liver through the use of hepatocyte-specific beta-catenin conditional knockout (KO) mice. SMP30 is a critical enzyme for the synthesis of ascorbic acid in murine hepatocytes, and its loss led to lower serum ascorbate levels in KO. KO hepatocytes displayed massive apoptosis in culture, which was blocked by addition of ascorbate to culture media. Additionally, apoptosis in HepG2 cells due to regucalcin knockdown was rescued by anti-oxidants. Thus, one mechanism of how beta-catenin regulates hepatocyte redox state and survival is through the control of regucalcin expression. KO livers displayed a basal increase in number of apoptotic hepatocytes. We explored the susceptibility of KO and wildtype (WT) controls to activation of the TNF-alpha mediated apoptotic pathway. Paradoxically, KO mice are refractory to D-galactosamine (GalN)/LPS, Actinomycin D (ActD)/LPS and GalN/TNF-alpha treatments showing lower morbidity than WT. NF-kappaB, a major pro-survival factor and its transcriptional targets were increased in KO basally and after injury due to lack of beta-catenin-p65 association, presence of increased basal inflammation and oxidative stress and increased TLR4 expression in KO livers. Additionally, p65 activation occurred earlier in KO than WT after LPS stimulation. Thus, paradoxical protection from TNF-alpha-mediated apoptosis in KOs occurs owing to pre-existing NF-kappaB activation that 'primes' the liver for protection against exogenous insult. Thus, we have identified beta-catenin as a pleiotropic factor regulating cell proliferation, cellular redox state and cell survival through specific genetic targets and protein-protein interactions. These findings have broad implications in acute and chronic hepatic diseases.

The generation of the pro-inflammatory cytokines IL-6 and TNF-α by macrophages recruited to adipose tissue facilitates obesity-induced inflammation resulting in insulin resistance and type 2 diabetes (T2D). Increased adipose tissue is associated with inflammation and expression of acute phase response (APR) proteins secreted by the liver. Proper homeostasis of the liver is regulated by IL-6-depdendent expression of Hepatocyte Growth Factor (HGF) upon cleavage to its active form (aHGF) by the urokinase-type plasminogen activator (uPA). Plasminogen Activator Inhibitor Type-1 (PAI-1) is a pro-thrombotic APR protein known to inhibit the function of uPA; however, since HGFs activation, interaction and signaling through its receptor, MET are dependent upon uPA, PAI-1 is also capable of regulating the function of hepatic HGF. In vitro data demonstrates that aHGF significantly suppressed IL-6 production by macrophages stimulated with LPS via an increase in phosphorylation of GSK3β, rendering it inactive. Phosphorylated GSK3β correlated with increased retention of the phosphorylated NF-κB p65 subunit in the cytoplasm and an enhanced interaction between CBP and phosphorylated CREB resulting in IL-10 cytokine production. These changes were a direct result of signaling through MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. It is known that obese T2D patients present with an accumulation of PAI-1, which we hypothesize, results in the inactivation of HGF. The loss of HGF-MET signaling results in increased active GSK3β and the progression to unchecked inflammation and disease progression. In vivo studies using male, C57BL6 mice on a high fat diet alongside control fed mice demonstrates move severe hepatic steatosis in obese mice at 44 weeks compared to control. Steatosis coincided with the decrease in aHGF and elevated levels of PAI-1 protein. These results demonstrate that elevated levels of PAI-1 inhibit aHGF, leading to unresolved chronic inflammation in obesity and T2D.

Podocytes are highly differentiated glomerular epithelial cells that play an essential role in the establishment of the glomerular filtration barrier, a structural apparatus that selectively restricts the filtration of different macromolecules in the blood stream on the basis of their sizes, shape and charge. Podocyte dysfunction, one of the major causes of proteinuria, is of pathogenetic and prognostic significance in human glomerular disease. My study is focused on the investigation of novel pathways leading to podocyte dysfunction and proteinuria. In the first part, immunoblotting and quantitative reverse transcriptase PCR (RT-PCR) were used to demonstrate that LIM and senescent cell antigen-like domains 1 (PINCH1) is induced and undergoes nuclear translocation in podocytes after transforming growth factor, beta 1 (TGF-beta1) treatment. Bioinformatics analysis revealed the putative nuclear export signal/nuclear localization signal (NES/NLS) at the PINCH1 C-terminus which is required for its nuclear translocation. Immunoprecipitation and GST pull-down assay identified the interaction between PINCH1 and Wilms tumor 1 (WT1) which led to suppression of the WT1-mediated podocalyxin gene expression. In vivo, PINCH1 also underwent nuclear translocation and interacted with WT1 after TGF-beta1 stimulation. Our data identifies nuclear transcription factor WT1 as a novel binding partner for PINCH1, and provides novel insight into the mechanism of podocyte dysfunction under pathological conditions. In the second part, RT-PCR results revealed that treatment with TGF-beta1 induced gene expression of several wingless-type MMTV integration site family members (Wnts), predominantly Wnt1, and activated beta-catenin in mouse podocytes. Wnt antagonist Dickkopf-1 (DKK1) blocked TGF-beta1-induced beta-catenin activation and preserved nephrin expression. In vivo, ectopic expression of constitutively active TGF-beta1 induced Wnt1 expression, activated glomerular beta-catenin, upregulated its downstream target genes, and led to podocyte injury and proteinuria. Consistently, concomitant expression of DKK1 gene abolished beta-catenin activation in mouse glomeruli, inhibited TGF-beta1-triggered Wnt/beta-catenin target genes, and ameliorated proteinuria. These results establish a role for Wnt/beta-catenin signaling in the pathogenesis of podocyte injury and also suggest that this signaling pathway could be exploited as a therapeutic target for the treatment of proteinuric kidney diseases.