Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus.

Previously, using FREP-MS, we identified a protein complex including eight proteins that specifically bind to the functional SNP (fSNP) rs6032664 at a CD40 locus associated with autoimmune diseases. Among these eight proteins, four are ribosomal proteins RPL26, RPL4, RPL8, and RPS9 that normally make up the ribosomal subunits involved in the cellular process of protein translation. So far, no publication has shown these ribosomal proteins function as transcriptional regulators. In this work, we demonstrate that four ribosomal proteins: RPL26, RPL4, RPL8, and RPS9 are bona fide CD40 transcriptional regulators via binding to rs6032664. In addition, we show that suppression of CD40 expression by RPL26 RNAi knockdown inactivates NF-κB p65 by dephosphorylation via NF-κB signaling pathway in fibroblast-like synoviocytes (FLS), which further reduces the transcription of disease-associated risk genes such as STAT4, CD86, TRAF1 and ICAM1 as the direct targets of NF-κB p65. Based on these findings, a disease-associated risk gene transcriptional regulation network (TRN) is generated, in which decreased expression of, at least, RPL26 results in the downregulation of risk genes: STAT4, CD86, TRAF1 and ICAM1, as well as the two proinflammatory cytokines: IL1ß and IL6 via CD40-induced NF-κB signaling. We believe that further characterization of this disease-associated TRN in the CD40-induced NF-κB signaling by identifying both the upstream and downstream regulators will potentially enable us to identify the best targets for drug development.

Ex vivo lung perfusion as a human platform for preclinical small molecule testing.

The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.

IL-4 Induces IL17Rb Gene Transcription in Monocytic Cells with Coordinate Autocrine IL-25 Signaling.

IL-25 and IL-4 signaling in the setting of infection or allergic responses can drive Type 2 inflammation. IL-25 requires the IL-17 receptor B (IL-17Rb) to mediate signaling through nuclear factor κ B (NF-κB) transcriptional activation. Despite the known coexistence of these two cytokines in the Type 2 inflammatory environment, collaborative signaling between the IL-4 and IL-25 axes is poorly explored. Here we demonstrate IL-4 induction of both IL-25 and IL-17Rb protein in human lung tissue culture, primary alveolar macrophages, and the THP-1 monocytic cell line. IL-4 treatment triggers gene transcription for both IL-25 and IL-17Rb but does not alter the receptor mRNA stability. Genetic antagonism of the IL-4 second messenger, signal transducer and activator of transcription 6 (STAT6), with small interfering RNA (siRNA) blunts IL-17Rb mRNA induction by IL-4. IL-25 induces signaling through the canonical NF-κB pathway, and STAT6 or NF-κB signaling inhibitors prevent IL-17Rb expression. Blockade of IL-25 with monoclonal antibody suppresses NF-κB activation after IL-4 treatment, and IL-4-mediated induction of IL-17Rb is suppressed by IL-25 siRNA. IL-25 and IL-17Rb promoter regions harbor putative NF-κB and STAT6 consensus sites, and chromatin immunoprecipitation identified these transcription factors in complex with the IL-17Rb 5' untranslated region. In bronchoalveolar lavage RNA preparations, IL-25 and IL-17Rb mRNA transcripts are increased in asthmatics compared with healthy control subjects, and IL-25 transcript abundance correlates strongly with IL-4 mRNA levels. Thus, these results indicate that IL-4 signaling up-regulates the IL-25 axis in human monocytic cells, and that IL-25 may provide autocrine signals in monocytes and macrophages to sustain IL-17Rb expression and predispose to alternative activation.

Thymoquinone Promotes Pancreatic Cancer Cell Death and Reduction of Tumor Size through Combined Inhibition of Histone Deacetylation and Induction of Histone Acetylation.

Pancreatic ductal adenocarcinoma (PDAC) is virtually therapy-resistant. As noninvasive lesions progress to malignancy, the precursor period provides a window for cancer therapies that can interfere with neoplastic progression. Thymoquinone (Tq), a major bioactive component of essential oil from Nigella sativa's seeds, has demonstrated antineoplastic activities in multiple cancers. In this study, we investigated antineoplastic potential of Tq in human PDAC cell lines, AsPC-1 and MiaPaCa-2. Tq (10-50 µM) inhibited cell viability and proliferation and caused partial G2 cycle arrest in dose-dependent manner in both cell lines. Cells accumulated in subG0/G1 phase, indicating apoptosis. This was associated with upregulation of p53 and downregulation of Bcl-2. Independently of p53, Tq increased p21 mRNA expression 12-fold. Tq also induced H4 acetylation (lysine 12) and downregulated HDACs activity, reducing expression of HDACs 1, 2, and 3 by 40-60%. In vivo, Tq significantly reduced tumor size in 67% of established tumor xenografts (P < 0.05), along with increased H4 acetylation and reduced HDACs expression. Our results showed that Tq mediated posttranslational modification of histone acetylation, inhibited HDACs expression, and induced proapoptotic signaling pathways. These molecular targets demonstrate rationale for using Tq as a promising antineoplastic agent to prevent postoperative cancer recurrence and to prolong survival of PDAC patients after surgical resection.

The Human IL-22 Receptor Is Regulated through the Action of the Novel E3 Ligase Subunit FBXW12, Which Functions as an Epithelial Growth Suppressor.

Interleukin- (IL-) 22 signaling is protective in animal models of pneumonia and bacteremia by Klebsiella pneumoniae and mediates tissue recovery from influenza and Staph aureus infection. We recently described processing of mouse lung epithelial IL-22 receptor (IL-22R) by ubiquitination on the intracellular C-terminal. To identify cellular factors that regulate human IL-22R, we screened receptor abundance while overexpressing constituents of the ubiquitin system and identify that IL-22R can be shuttled for degradation by multiple previously uncharacterized F-box protein E3 ligase subunits. We observe that in human cells IL-22R is destabilized by FBXW12. FBXW12 causes depletion of endogenous and plasmid-derived IL-22R in lung epithelia, binds the E3 ligase constituent Skp-1, and facilitates ubiquitination of IL-22R in vitro. FBXW12 knockdown with shRNA increases IL-22R abundance and STAT3 phosphorylation in response to IL-22 cytokine treatment. FBXW12 shRNA increases human epithelial cell growth and cell cycle progression with enhanced constitutive activity of map kinases JNK and ERK. These findings indicate that the heretofore-undescribed protein FBXW12 functions as an E3 ligase constituent to ubiquitinate and degrade IL-22R and that therapeutic FBXW12 inhibition may enhance IL-22 signaling and bolster mucosal host defense and infection containment.

Serum monocyte chemoattractant protein-1 in pancreatic cancer.

Background/Aims. Pancreatic ductal adenocarcinoma (PDA) has etiological association with chronic inflammation. Elevated circulating levels of inflammatory mediators, such as monocyte chemoattractant protein-1 (MCP-1), are found in obese individuals. We hypothesized that serum MCP-1 levels are elevated in obese PDA patients. Methods. ELISA was used to analyze MCP-1 serum levels in PDA (n = 62) and intraductal papillary mucinous neoplasms (IPMN) (n = 27). Recursive partitioning statistical analysis investigated the relationship between log MCP-1 and clinicopathological parameters. Results. Log MCP-1 values were significantly (P < 0.05) elevated in patients with BMI ≥ 37.5. In patients with BMI < 37.5, average log MCP-1 values were significantly elevated in PDA patients when compared to IPMN patients. Within the IPMN group, higher log MCP-1 levels correlated with increased age. Recursive partitioning analysis of IPMN versus PDA revealed a strategy of predicting characteristics of patients who are more likely to have cancer. This strategy utilizes log MCP-1 as the primary factor and also utilizes smoking status, gender, and age. Conclusion. MCP-1 is a promising biomarker in pancreatic cancer. The potential of using MCP-1 to distinguish PDA from IPMN patients must be studied in larger populations to validate and demonstrate its eventual clinical utility.

Angiotensin II type 2 receptor blockade inhibits fatty acid synthase production through activation of AMP-activated protein kinase in pancreatic cancer cells.

BACKGROUND: The lipogenesis-promoting enzyme fatty acid synthase is highly expressed in pancreatic ductal adenocarcinoma. Angiotensin II, which is the principal hormone of the renin angiotensin system, is generated actively in the pancreas and has been shown to increase the expression of fatty acid synthase. The angiotensin II type 2 receptor has been proposed to play an important role in lipogenesis and fat deposition. In this study, we explored the potential role of the angiotensin II type 2 receptor in fatty acid synthase regulation in pancreatic ductal adenocarcinoma cells, and we evaluated the mechanisms involved. METHODS: Fatty acid synthase messenger RNA and protein in pancreatic ductal adenocarcinoma cell lines treated with or without angiotensin II (10(-6) to 10(-8) mol/L) in the presence or absence of the angiotensin II type 2 receptor blocker PD123319 (10(-4) to 10(-6) mol/L) were analyzed by real-time polymerase chain reaction and Western blotting. The total-AMP-activated protein kinase and phospho-AMP-activated protein kinase, total-acetyl CoA carboxylase and phospho-acetyl CoA carboxylase, and LKB1/STK11 were analyzed by Western immunoblotting. The tissue localization of the angiotensin II type 2 receptor was examined by immunohistochemistry in invasive pancreatic ductal adenocarcinoma lesions and matching normal tissue. RESULTS: Angiotensin II type 2 receptor treatment increased fatty acid synthase expression and promoter activity in significantly pancreatic ductal adenocarcinoma cells; these effects were blocked significantly in the presence of PD123319. Interestingly, angiotensin II also induced angiotensin II type 2 receptor expression in pancreatic ductal adenocarcinoma cells. PD123319, C75, and AICAR decreased fatty acid synthase protein levels, but only PD123319 increased LKB1/STK11 levels. All 3 agents activated AMP-activated protein kinase differentially and inhibited acetyl CoA carboxylase. Angiotensin II type 2 receptor messenger RNA levels were upregulated significantly in 20 of the 25 neoplastic tissues examined (80%) when compared with matching controls. Angiotensin II type 2 receptor protein was localized in the malignant ducts and in the stromal cells. CONCLUSION: Our data demonstrate a previously unknown involvement of the angiotensin II type 2 receptor in pancreatic ductal adenocarcinoma cell fatty acid synthesis and suggest that its blockade has potential as a novel chemopreventive and antilipogenic mechanism for human pancreatic ductal adenocarcinoma through the activation of AMP-activated protein kinase, which could have detrimental effects on cancer cell survival.

Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. METHODS: We analyzed paired PDA-normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA. RESULTS: COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. CONCLUSION: We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.

Expression and regulation of nicotine receptor and osteopontin isoforms in human pancreatic ductal adenocarcinoma.

Osteopontin (OPN) is a secreted phospho-protein that confers on cancer cells a migratory phenotype. We have recently shown that nicotine, a risk factor in pancreatic ductal adenocarcinoma (PDA), induces an alpha7-nicotine acetylcholine receptor (α7-nAChR)-mediated increase of OPN in PDA cells. In this study, we tested nicotine's effect on the expression of OPN splice variants (OPNa, b, c) in PDA cells. We also analyzed the correlation between patients' smoking history with OPN and α7-nAChR levels. RT-PCR and UV-light-illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and PDA cells treated with or without nicotine (3-300 nM). Localization of total OPN, OPNc and α7-nAChR was analyzed by immunohistochemistry, and their mRNA tissue expression levels were correlated with the patients' smoking history. PDA cells expressed varying levels of OPNa, OPNb, and α7-nAChR. Nicotine treatment selectively induced denovo expression of OPNc and increased α7-nAChR expression levels. In PDA tissue, OPNc was found in 87% of lesions, of which 73% were smokers. OPNc and total OPN levels were correlated in the tissue from patients with invasive PDA. Nicotine receptor was expressed in the invasive and premalignant lesions without clear correlation with smoking history. We show here for the first time that α7-nAChR is expressed in PDA cells and tissues and is regulated by nicotine in PDA cells. This, together with our previous findings that α7-nAChR mediates the metastatic effects of nicotine in PDA, suggest that combined targeting of α7-nAChR and OPNc could be a valid novel therapeutic strategy for invasive PDA, especially in the smoking population.

Involvement of osteopontin in the matrix-degrading and proangiogenic changes mediated by nicotine in pancreatic cancer cells.

BACKGROUND: Substantial evidence indicates that exposure to cigarette smoke is associated with an elevated risk of pancreatic ductal adenocarcinoma (PDA). However, the mechanisms underlying the effects of nicotine on the development or progression of PDA remain to be investigated. Previously, we showed that nicotine promotes the expression of osteopontin c (OPNc), an isoform of OPN protein that confers on cancer cells a migratory phenotype. In this study, we explored the potential prometastatic role of nicotine in PDA through studying its effect on the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) and evaluated the role of OPN in mediating these effects. MATERIALS AND METHODS: MMP-9 and VEGF mRNA and protein were analyzed in PDA cells treated with or without nicotine (3-300 nM). Transient transfection and luciferase-labeled promoter studies evaluated the effects of OPNc and OPN protein on the transcription and translation of MMP-9 and VEGF. Real-time PCR and immunohistochemistry were used to analyze the mRNA expression levels and localization of OPN, MMP-9, and VEGF proteins in matched invasive human PDA and surrounding nonmalignant tissues. RESULTS AND DISCUSSION: Nicotine significantly enhanced the expression of MMP-9 and VEGF mRNA and protein in PDA cells. Blocking OPN with siRNA or OPN antibody prevented the nicotine-mediated increase of both MMP-9 and VEGF. Transient transfection of OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (p < 0.05) increased MMP-9 and VEGF mRNA expression levels and induced their promoter activities. In invasive PDA lesions, MMP-9 mRNA levels were significantly (p < 0.005) higher in smokers vs. nonsmokers. VEGF protein co-localized with MMP-9 and OPN in the malignant ducts and correlated well with their higher levels in invasive PDA lesions. CONCLUSIONS: Our data show for the first time that cigarette smoking and nicotine may contribute to PDA metastasis through inducing MMP-9 and VEGF and suggest that OPN plays a central role in mediating these effects. The presence of OPN as a downstream effector of nicotine that is capable of mediating its prometastatic effects in PDA cells is novel and could provide a unique therapeutic target to control pancreatic cancer aggressiveness, especially in the cigarette-smoking population.

Induction of monocyte chemoattractant protein-1 by nicotine in pancreatic ductal adenocarcinoma cells: role of osteopontin.

BACKGROUND: Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA). We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein (MCP)-1 and evaluated the role of OPN in mediating these effects. METHODS: MCP-1 mRNA and protein in PDA cells treated with or without nicotine (3-300 nmol/L) or OPN (0.15-15 nmol/L) were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Luciferase-labeled promoter studies evaluated the effects of nicotine and OPN on MCP-1 transcription. Intracellular and tissue colocalization of OPN and MCP-1 were examined by immunofluorescence and immunohistochemistry. RESULTS: Nicotine treatment significantly increased MCP-1 expression in PDA cells. Interestingly, blocking OPN with siRNA or OPN antibody abolished these effects. Transient transfection of the OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P < .05) increased MCP-1 mRNA and protein and induced its promoter activity. MCP-1 was found in 60% of invasive PDA lesions, of whom 66% were smokers. MCP-1 colocalized with OPN in PDA cells and in the malignant ducts, and correlated well with higher expression levels of OPN in the tissue from patients with invasive PDA. CONCLUSION: Our data suggest that cigarette smoking and nicotine may contribute to PDA inflammation by inducing MCP-1 and provide a novel insight into a unique role for OPN in mediating these effects.

Blocking angiotensin II Type 1 receptor triggers apoptotic cell death in human pancreatic cancer cells.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with an annual mortality rate close to its annual incidence. We recently demonstrated that angiotensin II (AngII) type 1 receptor (AT1R) might be involved in PDA angiogenesis. This study evaluated the antiproliferative and proapoptotic effects of an AT1R blocker, losartan, in PDA cells with different p53 mutation status. METHODS: Cell cycle was analyzed by flow cytometric analysis of DNA content; apoptosis by annexin V-fluorescein isothiocyanate (V-FITC) and terminal deoxytransferase (TdT)-mediated dUTP nick-end labeling staining; messenger RNA and protein by real-time polymerase chain reaction and Western blotting; caspase-3 activity by colorimetric assay; and promoter activity by luciferase assay. RESULTS: Losartan dose-dependently decreased cell survival and increased their preG1 accumulation. It also increased p53, p21, p27, and Bax and reduced Bcl-2 and Bcl-xl expression. In wtp53 cells, losartan increased p53 transcription and activated caspase-3 in both cell lines. However, its proapoptotic effects in mtp53 cells were mainly caspase-3-dependent. CONCLUSION: Our data describe the involvement of AT1R in PDA cell apoptotic machinery and provide the first evidences that losartan stimulates the proapoptotic signaling pathways regardless of the p53 mutation status. As loss of p53 function is frequently observed in PDA patients, our data suggest AT1R blockade as a novel therapeutic strategy to control PDA growth.

Angiotensin II regulates the expression of monocyte chemoattractant protein-1 in pancreatic cancer cells.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers with an overall median survival of less than 9 months and a 5-year survival rate of less than 5%. Increasing evidence indicates that inflammation facilitates PDA growth. DISCUSSION: Angiotensin II (AngII), the principal hormone of the renin-angiotensin system, is actively generated in the pancreas and has been proposed as a key mediator of inflammation. Monocyte chemoattractant protein (MCP)-1 is a chemokine that plays an important role in the recruitment of mononuclear cells into sites of inflammation. In this study, we investigated the potential proinflammatory role of AngII in PDA through studying its effect on MCP-1. AngII significantly increased the expression of MCP-1 mRNA and protein in PDA cells and induced its promoter activity. Constitutive and AngII-induced MCP-1 transcription was inhibited by an AngII type 1 receptor (AT1R) blocker, but was unchanged by an AT2R blocker. AngII activated the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 or c-Jun NH2-terminal mitogen-activated protein kinases. Inhibition of ERK1/2 activation reduced the AngII-induced MCP-1 synthesis. AngII induced the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB), an effect that was inhibited by AT1R blockade. Inhibition of NF-kappaB by pyrrolidine dithiocarbamate decreased the AngII-mediated increase in MCP-1 mRNA. Our data provide a novel insight into an AngII-initiated signal transduction pathway that regulates MCP-1 as a possible inflammatory mechanism in PDA and suggest that AngII blockade may regulate chemokine-induced signal transduction to prevent or reduce inflammation in PDA.

Anti-inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells.

BACKGROUND: Both hereditary and sporadic forms of chronic pancreatitis are associated with an increased risk of developing pancreatic ductal adenocarcinoma (PDA). Inflammation has been identified as a significant factor in the development of solid tumour malignancies. We have recently shown that thymoquinone (Tq), the major constituent of Nigella sativa oil extract, induced apoptosis and inhibited proliferation in PDA cells. Tq also increased p21 WAF1 expression, inhibited histone deacetylase (HDAC) activity, and induced histone hyperacetylation. HDAC inhibitors have been shown to ameliorate inflammation-associated cancer. In this study, we evaluated the anti-inflammatory potential of Tq in PDA cells in comparison with that of a specific HDAC inhibitor, trichostatin A (TSA). METHODS: PDA cells were treated with or without Tq (25-75 microM), with or without pre-treatment of tumour necrosis factor (TNF)-alpha (25 ng/ml). The effect of Tq on the expression of different proinflammatory cytokines and chemokines was analysed by real-time polymerase chain reaction (PCR). Luciferase-labelled promoter studies evaluated the effect of Tq on the transcription of monocyte chemoattractant protein-1 (MCP-1) and nuclear factor-kappaB (NF-kappaB). The effect of Tq on the constitutive and TNF-alpha-induced activation and nuclear translocation of NF-kappaB was examined by ELISA and immunohistochemistry. RESULTS: Tq dose- and time-dependently significantly reduced PDA cell synthesis of MCP-1, TNF-alpha, interleukin (IL)-1beta and Cox-2. At 24 h, Tq almost completely abolished the expression of these cytokines, whereas TSA had a less dramatic effect. Tq, but not TSA, significantly and dose-dependently reduced the intrinsic activity of the MCP-1 promoter. Tq also inhibited the constitutive and TNF-alpha-mediated activation of NF-kappaB in PDA cells and reduced the transport of NF-kappaB from the cytosol to the nucleus. CONCLUSIONS: Our data demonstrate previously undescribed anti-inflammatory activities of Tq in PDA cells, which are paralleled by inhibition of NF-kappaB. Tq as a novel inhibitor of proinflammatory pathways provides a promising strategy that combines anti-inflammatory and proapoptotic modes of action.

Expression of a prometastatic splice variant of osteopontin, OPNC, in human pancreatic ductal adenocarcinoma.

BACKGROUND: Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype. We demonstrated recently that nicotine, a major risk factor in pancreatic ductal adenocarcinoma (PDA), increases OPN expression in PDA cells. An OPN splice variant, OPNc, supports anchorage independence and maybe the most potent OPN isoform to convey metastatic behavior. In this study, we tested the effect of nicotine on OPNc expression and analyzed the correlation between total OPN/OPNc levels and patients' smoking history. METHODS: Real-time polymerase chain reaction and ultraviolet light illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and in PDA cells treated with or without nicotine (3-300 nmol/L). OPN and OPNc were localized by immunohistochemistry, and an enzyme-linked immunosorbent assay was used to analyze OPN serum levels. RESULTS: Nicotine treatment of PDA cells selectively induced de novo expression of OPNc. OPNc was found in 87% of invasive PDA lesions, of which 73% were found in smokers. The levels of OPNc correlated well with higher expression levels of total OPN in the tissue and serum from patients with invasive PDA. CONCLUSION: Our data suggest that smoking and nicotine may contribute to PDA metastatic potential through promoting OPNc expression. Although the direct role of OPNc in PDA progression is not defined, OPNc may have value as a diagnostic and prognostic marker, especially in invasive PDA.

Induction of osteopontin expression by nicotine and cigarette smoke in the pancreas and pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with etiological association with cigarette smoking. Nicotine, an important component of cigarettes, exists at high concentrations in the bloodstream of smokers. Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival, proliferation, invasion, and metastasis. Here, we investigated the potential molecular basis of nicotine's role in PDA through studying its effect on OPN. Nicotine significantly (p < 0.02) increased OPN mRNA and protein secretion in PDA cells through activation of the OPN gene promoter. The OPN mRNA induction was inhibited by the nicotinic acetylcholine receptor antagonist, mechamylamine. Further, the tyrosine kinase inhibitor genistein inhibited the nicotine-mediated induction of OPN, suggesting that mitogen activated protein kinase signaling mechanism is involved. Nicotine activated the phosphorylation of ERK1/2, but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the nicotine-induced OPN synthesis. Rats exposed to cigarette smoke showed a dose-dependent increase in pancreatic OPN that paralleled the rise of pancreatic and plasma nicotine levels. Analysis of cancer tissue from invasive PDA patients, the majority of whom were smokers, showed the presence of significant amounts of OPN in the malignant ducts and the surrounding pancreatic acini. Our data suggest that nicotine may contribute to PDA pathogenesis through upregulation of OPN. They provide the first insight into a nicotine-initiated signal transduction pathway that regulates OPN as a possible tumorigenic mechanism in PDA.

Expression and regulation of osteopontin in type 1 diabetes.

Osteopontin (OPN) is a secreted acidic phosphoprotein that is involved in many inflammatory and immune-modulating disorders. We previously demonstrated that OPN is a novel islet protein and a pro survival factor that may serve as an intrinsic feedback regulator of nitric oxide signaling in ß-cells. Here, we investigated the endogenous expression of pancreatic OPN in non obese diabetic (NOD) mice and explored its regulation in the islets and b-cells. High levels of pancreatic OPN mRNA and protein were seen in the prediabetic NOD mice pancreata. The temporal pattern of OPN expression inversely correlated with progression of insulitis and ß-cell destruction. Immunostaining of pancreatic serial sections showed co localization of OPN with most of the islet hormones. Next we investigated the regulation of OPN in the islets and ß-cells. Naturally occurring early upregulation of OPN transcription was seen after exposure of native normoglycemic NOD islets and ß-cells to a high-dose combination of IL-1ß, TNF-α and IFN-γ. To distinguish between the effect of cytokines and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with cytokines or glucose. Cytokines induced upregulation of OPN promoter activity within one hour, while glucose induced a dose-dependent upregulation of OPN promoter activity after 24 hrs. Long-term exposures to cytokines or glucose reduced OPN expression and promoter activity. Our data provide the first observations into the presence of a positive intrinsic mechanism that regulates pancreatic OPN expression. Based upon previous studies that support a protective role of OPN in the islets, our data suggest that exhaustion of this local OPN system is implicated in the associated loss of endogenous islet protection and progression of the destructive insulitis and diabetes severity in the NOD mouse model.

Angiotensin II induces vascular endothelial growth factor in pancreatic cancer cells through an angiotensin II type 1 receptor and ERK1/2 signaling.

Vascular endothelial growth factor (VEGF) is a crucial pro-angiogenic component in pancreatic ductal adenocarcinoma (PDA), and its high expression levels have been correlated with poor prognosis and early postoperative recurrence. We have recently shown that high levels of angiotensin II (AngII) type 1 receptor (AT1R) correlate and colocalize with VEGF in invasive PDA and that AngII induces VEGF expression in PDA cell lines. In this study, we explored the signaling mechanisms involved in the AngII-mediated VEGF induction and correlated AT1R and VEGF expression in noninvasive precursor lesions. An AT1R antagonist significantly (p<0.05) inhibited the AngII-mediated induction of VEGF messenger RNA and protein in all PDA cell lines. AngII-VEGF induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a mitogen-activated protein kinase signaling mechanism. AngII activated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the AngII-induced VEGF synthesis. Immunohistochemical analysis of precursor lesions showed increased expression of AT1R in most ductal cells undergoing metaplasia. Pancreatic intraepithelial neoplasms showed more intense AT1R staining when compared to intraductal papillary mucinous neoplasms, which showed heterogeneous immunoreactivity. VEGF followed the same distribution pattern of AT1R in both lesions. AT1R expression in the premalignant pancreatic lesions suggests its involvement in tumor progression and angiogenesis. Our mechanistic findings provide the first insight into an AngII-initiated signaling pathway that regulates PDA angiogenesis. An AT1R-mediated VEGF induction suggests the possibility of AT1R blockade as a novel therapeutic strategy to control angiogenesis in PDA.

Antihypertensives as novel antineoplastics: angiotensin-I-converting enzyme inhibitors and angiotensin II type 1 receptor blockers in pancreatic ductal adenocarcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a crucial proangiogenic component in pancreatic ductal adenocarcinoma (PDA), and its high expression levels have been correlated with poor prognosis and early postoperative recurrence. Angiotensin II (AngII), which has been shown to increase VEGF production in a variety of cancers, is actively generated in the pancreas. We hypothesized that AngII plays a crucial role in PDA-associated angiogenesis. STUDY DESIGN: We analyzed the expression and localization of AngI converting enzyme (ACE) and AngII type 1 receptor (AT1R) in relation to VEGF in matched invasive human PDA (n=25) and surrounding nonmalignant tissues using real-time polymerase chain reaction, Western immunoblotting, and immunohistochemistry. VEGF levels in conditioned media of HS766T and PK9 PDA cells treated with or without AngII (10(-7) mol/L) were measured by ELISA. The effects of an AT1R blocker (losartan) and an ACE inhibitor (captopril) on VEGF production and cellular proliferation were also examined. RESULTS: ACE and AT1R mRNA and protein levels were significantly upregulated in 19 of the 25 neoplastic tissues examined (approximately 75%), when compared with matching controls. VEGF expression was significantly higher in tissues that expressed high levels of AT1R and ACE (n=19), compared with low levels (n=4) or negative (n=2) cases. ACE protein collocalized with AT1R and VEGF in the malignant ducts and in the stromal cells. Addition of AngII significantly enhanced VEGF mRNA production and protein secretion, an effect that was prevented when cells were preincubated with captopril or losartan. Blocking endogenous AngII by captopril or losartan significantly suppressed cell proliferation. CONCLUSIONS: Both ACE and AT1R are functionally expressed in PDA and may be involved in tumor angiogenesis. Because AT1R blockers and ACE inhibitors are already widely used clinically, they may represent a potential novel and promising strategy for controlling angiogenesis, prevention of metastasis, and prolongation of survival in patients with primary or metastatic PDA.

Induction of monocyte chemoattractant protein-1 expression by angiotensin II in the pancreatic islets and beta-cells.

Angiotensin II (AngII), the principal hormone of the renin-angiotensin system, is actively generated in the pancreas and has been suggested as a key mediator of inflammation. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of mononuclear cells into the pancreatic islets. In this study, we investigated the potential molecular basis for the role of AngII in islet inflammation through studying its effect on MCP-1. AngII significantly increased the expression of MCP-1 mRNA and protein in the RINm5F beta-cell line and activated MCP-1 promoter. AngII-MCP-1 mRNA induction was inhibited by an AngII type 1 receptor antagonist but was unchanged by an AngII type 2 receptor antagonist. AngII-MCP-1 induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a MAPK signaling mechanism. AngII activated the phosphorylation of ERK1/2 but not p38 or c-Jun NH(2)-terminal MAPKs. Inhibition of ERK1/2 activation reduced the AngII-induced MCP-1 synthesis. In nonobese diabetic mice pancreata, the temporal pattern of angiotensin-converting enzyme expression correlated well with progression of insulitis and beta-cell destruction. Immunostaining of pancreatic serial sections show colocalization of angiotensin-converting enzyme with MCP-1 in beta-cells in the islets. In freshly isolated islets from normoglycemic mice, AngII alone and in combination with IL-1beta elicited an inflammatory response by stimulation of MCP-1. Our data suggest a positive autocrine/paracrine action for the local pancreatic AngII-generating system during insulitis and provide the first insight into an AngII-initiated signal transduction pathway that regulates MCP-1 as a possible inflammatory mechanism in the islets.