MUTATIONS IN STAT3 ASSOCIATED WITH HUMAN HYPER IgE SYNDROME ENHANCE NFκB AND MAPK-MEDIATED GENE EXPRESSION

Hyper IgE syndrome (HIES) is a multisystem disorder resulting in bone and immune system abnormalities. It is associated with mutations in STAT3, which disrupt protein domains responsible for transcriptional function. Patients with HIES display osteoporosis and enhanced inflammatory cytokine production similar to hematopoietic Stat3-deficient mice. Since osteoclast and inflammatory cytokine genes are NFκB targets, these observations indicate a possible deregulation of NFκB signaling in both mice and humans with STAT3-deficiency. Here, we sought to examine the role of STAT3 in the regulation of NFκB-mediated gene expression through analysis of three HIES STAT3 point mutations in both hematopoietic and non- hematopoietic cells. We found that IL-6-induced tyrosine phosphorylation of STAT3 was partially or completely abrogated by HIES mutations in the transactivation domain (V713L) or SH2 domain (V637M), respectively, in both hematopoietic and non- hematopoietic cells. By contrast, IL-6-induced tyrosine phosphorylation of an HIES mutant in the STAT3 DNA-binding domain (R382W) was intact. The R382W and V713L mutants significantly reduced IL-6-dependent STAT3 transcriptional activity in reporter gene assays. Moreover, the R382W and V637M mutants significantly diminished IL-6-responsive expression of the endogenous STAT3 target gene, Socs3, as assessed by quantitative real-time PCR (qPCR) in the RAW macrophage cell line. These observations indicate the HIES mutants dominantly suppress the transcriptional activity of wild type STAT3, albeit to varying degrees. All three HIES mutants enhanced LPS-induced expression of the NFκB target genes IL6 (IL-6), Cxcl10 (IP- 10), and Tnf (TNFα) in RAW cells, as indicated by qPCR. Furthermore, overexpression of wild type STAT3 in Stat3-deficient murine embryonic fibroblasts significantlyreduced LPS-stimulated expression of IL6, Cxcl10, and IL12p35. In addition, in aprimary murine osteoclast differentiation assay, a STAT3-specific SH2 domain inhibitor led to significantly increased levels of osteoclast-specific gene expression. These results suggest that STAT3 serves as a negative regulator of NFκB-mediated gene expression, and furthermore imply that STAT3 mutations associated with HIES contribute to the osteopenia and inflammation observed in HIES patients.

CHARACTERIZATION OF THE ROLE OF CARMA3 IN ENDOPLASMIC RETICULUM STRESS-INDUCED NF-κB ACTIVATION

Endoplasmic reticulum (ER) stress-induced inflammation plays an important role in the progression of many diseases, such as type II diabetes, insulin resistance, cancers, and so on. NF-κB is believed to be a central regulator of ER stress-induced inflammation. However, studies on how ER stress induces NF-κB activation are limited and, in some cases, controversial. In the present study, we utilized two commonly used ER stress inducers, thapsigargin and tunicamycin, to study the mechanism. We found that two caspase-recruitment domain (CARD)-containing proteins, CARMA3 and BCL10, play a crucial roles on ER stress-induced NF-κB activation by regulating IκBα kinase activity. Consistently, we observed that a physiological ER stress inducer, hypoxia, could activate NF-κB in a CARMA3-dependent manner. Additionally, we showed that the activation of the UPR signaling pathways were intact in both CARMA3- and BCL10-deficient cells under ER stress. Together, this study provides insight into the mechanism of how ER stress induces NF-κB activation. It allows us to better understand ER stress-induced inflammation and develop the corresponding therapeutic interference to treat diseases

PML tumor suppressor potentiates cell death through inhibition of the NF -kappa B survival pathway

The promyelocytic leukemia protein PML is a growth suppressor essential for induction of apoptosis by diverse apoptotic stimuli. The mechanism by which PML regulates cell death remains unclear. In this study we found that ectopic expression of PML potentiates cell death in the TNFα-resistant tumor line U2OS and significantly sensitized these cells to apoptosis induced by TNFα in a p53-independent manner. Our study demonstrated that both PML and PML/TNFα-induced cell death are associated with DNA fragmentation, activation of caspase-3, -7, -8, and degradation of DFF/ICAD. Furthermore, we found that PML-induced and PML/TNFα-induced cell death could be blocked by the caspase-8 inhibitors crmA and c-FLIP, but not by Bcl-2, the inhibitor of mitochondria-mediated apoptotic pathway. These findings indicate that this cell death event is initiated through the death receptor-dependent apoptosis pathway. Our study further showed that PML recruits NF-kappa B (NF-κB) to the PML nuclear body, blocks NF-κB binding to its cognate enhancer, and represses its transactivation function with the C-terminal region. Therefore PML inhibits the NF-κB survival pathway. Overexpression of NF-κB rescued cell death induced by PML and PML/TNFκ. These results imply that PML is a functional repressor of NF-κB. This notion was further supported by the finding that the PML−/− mouse embryo fibroblasts (MEFs) are more resistant than the wild-type MEFs to TNFκ-induced apoptosis. In conclusion, our studies convincingly demonstrated that PML potentiates cell death through inhibition of the NF-κB survival pathway. Activation of NF-κB frequently occurs during oncogenesis. Our study here suggests that a loss of PML function enhances the NF-κB survival pathway and this event may contribute to tumorigenesis. ^

Dysregulated CD40-NF-kappaB signaling in the pathophysiology of aggressive non -Hodgkin's lymphoma B cells

Non-Hodgkin's Lymphomas (NHL) are a group (>30) of important human lymphoid cancers that unlike other tumors today, are showing a marked increase in incidence. The lack of insight to the pathogenesis of B-cell NHL poses a significant problem in the early detection and effective treatment of these malignancies. This study shows that large B-cell lymphoma (LBCL) cells, the most common type of B-cell NHL (account for more than 30% of cases), have developed a novel mechanism for autonomous neoplastic B cell growth. We have identified that the key transcription factor NF-κB, is constitutively activated in LBCL cell lines and primary biopsy-derived LBCL cells, suggesting that they are autonomously activated, and do not require accessory T-cell signaling for cell growth and survival. Further studies have indicated that LBCL cells ectopically express an important T-cell associated co-mitogenic factor, CD154 (CD40 ligand), that is able to internally activate the CD401NF-κB pathway, through constitutive binding to its cognate receptor, CD40, on the lymphoma cell surface. CD40 activation triggers the formation of a “Signalosome” comprising virtually the entire canonical CD40/NF-κB signaling pathway that is anchored by CD40 in plasma membrane lipid rafts. The CD40 Signalosome is vulnerable to interdiction by antibody against CD40 that disrupts the Signalosome and induces cell death in the malignant cells. In addition to constitutive NF-κB activation, we have found that the nuclear factor of activated T cells (NFAT) transcription factor is also constitutively activated in LBCL cells. We have demonstrated that the constitutively active NFATc1 and c-rel members of the NFAT and NF-κB families of transcription factors, respectively, interact with each other, bind to the CD154 promoter, and synergistically activate CD154 gene transcription. Down-regulation of NFATc1 and c-rel with small interfering RNA inhibits CD154 gene transcription and lymphoma cell growth. Our findings suggest that continuous CD40 activation not only provides dysregulated proliferative stimuli for lymphoma cell growth and extended tumor cell survival, but also allows continuous regeneration of the CD40 ligand in the lymphoma cell and thereby recharges the system through a positive feedback mechanism. Targeting the CD40/NF-κB signaling pathway could provide potential therapeutic modalities for LBCL cells in the future. ^

beta-catenin interacts with and inhibits NF-kappaB in colon cancer

The β-catenin pathway plays an important role in the progression of colon cancer as well as many other cancer types. Almost all colorectal tumors show an upregulation of β-catenin activity either through mutations in the β-catenin regulator APC or through mutations in β-catenin itself. Upregulation of β-catenin leads to the transcription of many target genes involved in tumorigenesis. NF-κB is a transcription factor which activates many target genes, including both anti-apoptotic and pro-apoptotic molecules. Recently, it has been shown that GSK-3β, a negative regulator of β-catenin, is involved in the activation of NF-κB. However, the mechanism of this regulation of NF-κB by GSK-3β is unclear. As GSK-3β inhibits β-catenin we hypothesized that β-catenin may be responsible for the regulation of NF-κB by GSK-3β; i.e. β-catenin may inhibit NF-κB activity. In this study we show that β-catenin physically interacts with NF-κB leading to the inhibition of NF-κB transcriptional and DNA-binding activities. We also show that in colon cancer cells with high β-catenin expression there is a suppressed NF-κB activity and depletion of β-catenin increases NF-κB activity. Similarly, in colon cancer cells that have a low level of β-catenin NF-κB activity is high and introduction of β-catenin reduces NF-κB activity. Importantly, we show that this suppression of NF-κB by β-catenin leads to a reduction of NF-κB target gene Fas expression. Also Fas-mediated apoptosis is reduced in β-catenin overexpressing cells, which can be reversed upon depletion of β-catenin. Introduction of the NF-κB subunit p65 can restore Fas expression indicating that the effect of β-catenin on Fas is through NF-κB. Furthermore, β-catenin expression was found to inversely correlate with Fas expression in human colon and breast primary tumor tissues. As Fas downregulation is important for tumors to evade immune surveillance, β-catenin inhibition of NF-κB and Fas downregulation likely plays and important role for colon cancer progression. Additionally, we found that phosphoinositide 3-kinase plays a role in the regulation of β-catenin inhibition of NF-κB through the disruption of the β-catenin/NF-κB complex. This study provides a link between two important signal transduction pathways as well as another mechanism of β-catenin oncogenesis. ^

Molecular mechanisms of NF-kappaB activation in metastatic pancreatic cancer cells

Pancreatic adenocarcinoma is the fourth leading cause of adult cancer death in the United States. At the time of diagnosis, most patients with pancreatic cancer have advanced and metastatic disease, which makes most of the traditional therapeutic strategies are ineffective for pancreatic cancer. A better understanding of the molecular basis of pancreatic cancer will provide the approach to identify the new strategies for early diagnosis and treatment. NF-κB is a family of transcription factor that play important roles in immune response, cell growth, apoptosis, and tumor development. We have shown that NF-κB is constitutively activated in most human pancreatic tumor tissues and cell lines, but not in the normal tissues and HPV E6E7 gene-immortalized human pancreatic ductal epithelial cells (HPDE/E6E7). By infecting the pancreatic cancer cell line Aspc-1 with a replication defective retrovirus expressing phosphorylation-defective IκBα (IκBαM), the constitutive NF-κB activation is blocked. Subsequent injection of this Aspc-1/IκBαM cells into the pancreas of athymic nude mice showed that liver metastasis is suppressed by the blockade of NF-κB activation. Current studies showed that an autocrine mechanism accounts for the constitutive activation of NF-κB in metastatic human pancreatic cancer cell lines, but not in nonmetastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1α (IL-1α) was the primary cytokine secreted by these cells that activates NF-κB. Inhibition of IL-1α activity suppressed the constitutive activation of NF-κB and the expression of its downstream target gene, uPA, in metastatic pancreatic cancer cell lines. Even though IL-1α is one of the previously identified NF-κB downstream target genes, our results demonstrate that regulation of IL-1α expression is independent of NF-κB and primarily dependent on AP-1 activity, which is in part induced by overexpression of EGF receptors and activation of MAP kinases. In conclusion, our findings suggest a possible mechanism by which NF-κB is constitutively activated in metastatic human pancreatic cancer cells and a possible missing mechanistic links between inflammation and cancer. ^

The transcription factor nuclear factor kappa B (NFkappaB) maintains TRAIL resistance in human pancreatic cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF family of cytokines that induces apoptosis in a variety of tumor cells while sparing normal cells. However, many human cancer cell lines display resistance to TRAIL-induced apoptosis and the mechanisms contributing to resistance remain controversial. Previous studies have demonstrated that the dimeric transcription factor Nuclear Factor kappa B (NFκB) is constitutively active in a majority of human pancreatic cancer cell lines and primary tumors, and although its role in tumor progression remains unclear it has been suggested that NFκB contributes to TRAIL resistance. Based on this, I examined the effects of NFκB inhibitors on TRAIL sensitivity in a panel of nine pancreatic cancer cell lines. I show here that inhibitors of NFκB, including two inhibitors of the proteasome (bortezomib (Velcade™, PS-341) and NPI-0052), a small molecule inhibitor of IKK (PS1145), and a novel synthetic diterpene NIK inhibitor (NPI-1342) reverse TRAIL resistance in pancreatic cancer cell lines. Further analysis revealed that the expression of the anti-apoptosic proteins BclXL and XIAP was significantly decreased following exposure to these inhibitors alone and in combination with TRAIL. Additionally, treatment with NPI0052 and TRAIL significantly reduced tumor burden relative to the control tumors in an L3.6pl orthotopic pancreatic xenograft model. This was associated with a significant decrease in proliferation and an increase in caspase 3 and 8 cleavage. Combination therapy employing PS1145 or NPI-1342 in combination with TRAIL also resulted in a significant reduction in tumor burden compared to either agent alone in a Panc1 orthotopic xenograft model. My studies show that combination therapy with inhibitors of NFκB alone and TRAIL is effective in pre-clinical models of pancreatic cancer and suggests that the approach should be evaluated in patients. ^

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

CARMA3 serves as a critical mediator of NF-kappaB in multiple signal transduction pathways

The central dogma of molecular biology dictates that DNA is transcribed into RNA, which is later translated into protein. One of the early activators in this process is the transcription factor NF-κB. We have determined that an NF-κB inducer, CARMA3, is required for proper neural tube closure, similar to other NF-κB inducers. Using a genetic knockout of CARMA3, we demonstrated that it is required for Gαq-coupled GPCR-induced NF-κB activation. This is facilitated through a MAPK and IKK phosphorylation-independent mechanism, most likely by controlling NEMO-associated ubiquitination. We have also shown that CARMA3 is required for EGF and HRG-induced NF-κB activation. This activation requires the activity of both EGFR and HER2, as well as PKC. Again, we observed no defect in IKK phosphorylation, although we determined a clear defect in IKK activation. Finally, we have begun to determine the role of CARMA3 to both EGFR and HER2-induced tumorigenicity. By overexpressing a constitutive active mutant of HER2 in our CARMA3 WT and KO MEF cells, we have shown CARMA3 is important for HER2-driven soft agar colony growth. We have also shown that knockdown of endogenous CARMA3 in the EGFR-overexpressing A431 cell line abolishes EGF-induced NF-κB activation. These same cells have a dramatically reduced capacity to form colonies in soft agar as well. Using both mouse xenografts and a transgenic model of HER2-induced breast cancer, we have initiated studies which will help to determine the role of CARMA3 to in vivo tumorigenesis. Collectively, this work reveals novel roles for the CARMA3 protein in development, GPCR and EGFR/HER2 signaling. It also suggests that CARMA3 is involved in EGFR/HER2 mediated tumorigenesis, possibly indicating a novel therapeutic target for use in treatment of cancer. ^

The role of beta-arrestin 2 in lysophosphatidic acid-induced NF-kappaB activation

Lysophosphatidic acid (LPA) is a bioactive phospholipid and binds to its receptors, a family of G protein-coupled receptors (GPCR), which initiates multiple signaling cascades and leads to activation of several transcription factors, including NF-κB. NF-κB critically regulates numerous gene expressions, and is persistently active in many diseases. In our previous studies, we have demonstrated that LPA-induced NF-κB activation is dependent on a novel scaffold protein, CARMA3. However, how CARMA3 is recruited to receptor remains unknown. β-Arrestins are a family of proteins involved in desensitization of GPCR signaling. Additionally, β-arrestins function as signaling adaptor proteins, and mediate multiple signaling pathways. Therefore, we have hypothesized that β-arrestins may link CARMA3 to LPA receptors, and facilitate LPA-induced NF-κB activation. ^ Using β-arrestin-deficient MEFs, we found that β-arrestin 2, but not β-arrestin 1, was required for LPA-induced NF-κB activation. Also, we showed that the expression of NF-κB-dependent cytokines, such as interlukin-6, was impaired in β-arrestin 2-deficient MEFs. Mechanistically, we demonstrated the inducible association of endogenous β-arrestin 2 and CARMA3, and we found the CARD domain of CARMA3 interacted with 60-320 residues of β-arrestin 2. To understand why β-arrestin 2, but not β-arrestin 1, mediated NF-κB activation, we generated β-arrestin mutants. However, some mutants degraded quickly, and the rest did not rescue NF-κB activation in β-arrestin-deficient MEFs, though they had similar binding affinities with CARMA3. Therefore, it indicates that slight changes in residues may determine the different functions of β-arrestins. Moreover, we found β-arrestin 2 deficiency impaired LPA-induced IKK kinase activity, while it did not affect LPA-induced IKKα/β phosphorylation. ^ In summary, our results provide the genetic evidence that β-arrestin 2 serves as a positive regulator in NF-κB signaling pathway by connecting CARMA3 to LPA receptors. Additionally, we demonstrate that β-arrestin 2 is required for IKKα/β activation, but not for the inducible phosphorylation of IKKα/β. Because the signaling pathways around the membrane-proximal region of LPA receptors and GPCRs are quite conserved, our results also suggest a possible link between other GPCRs and CARMA3-mediated NF-κB activation. To fully define the role of β-arrestins in LPA-induced NF-κB signaling pathways will help to identify new drug targets for clinical therapeutics.^

Pathways leading to apoptosis resistance in a murine B -cell lymphoma cell system

The aberrant activation of signal transduction pathways has long been linked to uncontrolled cell proliferation and the development of cancer. The activity of one such signaling module, the Mitogen-Activated Protein Kinase (MAPK) pathway, has been implicated in several cancer types including pancreatic, breast, colon, and lymphoid malignancies. Interestingly, the activation of MAP-Kinase-Kinase-Kinase proteins often leads to the additional activation of NF-κB, a transcription factor that acts as a cell survival signal through its control of antiapoptotic genes. We have investigated the role of a specific dimer form of the NF-κB transcription factor family, NF-κB1 (p50) homodimers, in its control of the proto-oncogene, Bcl-2, and we have identified the MEK/ERK (MAPK) signaling cascade as a mediator of NF-κB1 activity. ^ Two murine B cell lymphoma cell lines were used for these studies: LY-as, an apoptosis proficient line with low Bcl-2 protein expression and no nuclear NF-κB activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. Experiments modulating p50 activity correlated the activation of p50 homodimers with Bcl-2 expression and additional gel shift experiments demonstrated that the Bcl-2 P1 promoter had NF-κB sites with which recombinant p50 was able to interact. In vitro transcription revealed that p50 enhanced the production of transcripts derived from the Bcl-2 P1 promoter. These data strongly suggest that Bcl-2 is a target gene for p50-mediated transcription and suggest that the activation of p50 homodimers contributes to the expression of Bcl-2 observed in LY-ar cells. ^ Studies of upstream MAPK pathways that could influence NF-κB activity demonstrated that LY-ar cells had phosphorylated ERK proteins while LY-as cells did not. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK, a reversal of nuclear p50 homodimer DNA binding, and a decrease in the amount of Bcl-2 protein expression. Similarly, the activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with TNFα, an IKK activator, did not change the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an IKK-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. ^ These data indicate that the activation of the MEK/ERK MAP-Kinase signaling pathway acts upstream of p50 homodimer activation and Bcl-2 expression in this B cell lymphoma cell system and suggest that the activation of MEK/ERK may be a key step in the progression of lymphoma to advanced-staged disease. Other researchers have used MEK inhibitors to inhibit cell growth and sensitize a number of tumors to chemotherapies. In light of our data, MEK inhibitors may additionally be useful clinically to radiosensitize cancers of lymphoid origin. ^

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.

AUTOIMMUNE RESPONSES TO ATHEROSCLEROTIC

Atherosclerosis is a chronic, complex arterial disease characterized by intimal lipid accumulation and inflammation. A unique lipid-binding molecule, namely cluster of differentiation 1d (CD1d), may impact atherosclerosis. Structurally, CD1d acts as a nonpolymorphic cell-surface receptor, resembling the major histocompatibility complex-I (MHC-I). While MHC-I restricts peptide antigen presentation to T cells, CD1d presents lipid antigens to T cells named CD1d-restrictedd T cells. Although increased expression of CD1d has been found in human plaques, the exact nature of CD1d-recognized lipids in atherosclerosis remains to be determined. Three groups of lipids may undergo oxidation in atherosclerosis producing atherogenic lipids: phospholipids, fatty acids, and cholesterol. The central hypothesis is that CD1d recognizes and present oxidative lipids to activate CD1d-restricted T cells, and trigger proinflammatory signal transduction In the first part of this study, oxidative phospholipids were identified and characterized as potential autoantigen for CD1d-restricted T cells. Derived from phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine by oxidization, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) is commonly found in atherosclerotic plaques. Upon stimulation with PGPC, spleen-derived CD1d-restricted T cells produced higher levels of cytokines and proliferated at higher rates than those without PGPC stimulation. CD1d deficiency compromised the PGPC-triggered T cell activation, suggesting that PGPC may function as a potentially novel autoantigen for T cells in atherosclerosis. In the second part of this study, CD1d-mediated proinflammatory signaling was evaluated in murine models. Enhanced CD1 expression occurred in spleens of db/db mice with hyperlipidemia. Tumor necrosis factor-alpha (TNF-α) was increased in db/db spleen, while TNF-α receptor expression augmented in the db/db murine heart, in comparison with those in normal mice. The nuclear factor-κ B (NF-κB) expression was enhanced in the db/db heart, whereas CD1d-null mice showed lower NF-κB, implying the involvement of CD1d in inflammation of the spleen and heart tissues in the mice with hyperlipidemia. The current study has identified PGPC as a novel lipid antigen recognized by CD1d-restricted T cells in atherosclerosis. The animal study has also provided evidence that CD1d regulates NF-κB-mediated proinflammatory signaling. Hence, CD1d-restricted T cell responses to autolipid antigen and mediated inflammatory signal may represent a new molecular pathway that triggers cardiovascular tissue injury in atherosclerosis and hyperlipidemia.

THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Transcriptional regulation of the human Fas (CD95) promoter

The coordination of the apoptotic program necessitates the timely expression of sensor, effector, and mediator molecules. Fas/CD95, a transmembrane receptor which tethers the cell-death machinery, triggers apoptosis to maintain immune homeostasis, tolerance, and surveillance. Dysregulation in Fas-mediated apoptosis, either from disproportionate expression or disruptions in the downstream signaling pathway, manifests in autoimmune disorders and certain malignant progression. ^ In this project, the transcriptional requirements underlying two modulators of Fas expression were investigated. In T-lymphocytes, activation results in potent Fas upregulation followed by an acquisition of sensitivity towards FasL-mediated apoptosis. Human fas promoter cloning and analysis have identified a cis-element critical for inducible Fas expression. EMSA studies using this region demonstrated a constitutive association with the transcription factor Sp1 and inducible NF-κB binding in response to activation. These interactions were mutually exclusive, as the rB/Sp1 element bound with recombinant Sp1 was readily displaced by increasing amounts of NF-κB p50. Thus, Fas upregulation by T-cell activation stimuli is dependent upon NF-κB binding at the fas promoter. ^ The capacity of Sp1 to direct basal Fas expression was examined through mutagenesis of several GC-rich regions within the core fas promoter. Reporter analysis of single or combinatorial mutant GC-box constructs revealed usage of a particular GC-element in moderating over 50% of basal fas transcription. Inducible expression was Sp1-independent, however, since activated Jurkat cells containing fas Sp1-mutant constructs retained equivalent reporter induction. Overall, a dual-level of transcriptional control exists in fas, where constitutive activity is monitored through Sp1 binding, whereas T-cell activation obligates NF κB transactivation. ^ In response to genotoxic damage, p53 modulates Fas levels partly by a transcription-dependent mechanism. Reconstitution of wild-type p53 in the hepatoma cell line Hep3B readily induced Fas transcription. Furthermore, fas promoter analysis identified an undescribed p53 responsive element which, when deleted, ablated p53-mediated reporter activity. Therefore, the pro-apoptotic function mediated by p53 is driven partially through the enhancement of Fas expression. ^ Altogether, events elicting Fas transcription may invoke single or overlapping mechanisms that converge at the level of promoter activity. Agents that enhance or attenuate these pathways may be therapeutically beneficial in modulating the expression and sensitivity towards Fas-dependent apoptosis. ^