The NF-κB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription.

BACKGROUND: Inducible nitric oxide synthase (iNOS) metabolizes L-arginine to produce nitric oxide (NO) which was originally identified in myeloid cells as a host defense mechanism against pathogens. Recent studies, however, have revealed that iNOS is often induced in tumor cells and myeloid cells in the tumor microenvironment. Compelling experimental data have shown that iNOS promotes tumor development in certain cellular context and suppresses tumor development in other cellular conditions. The molecular mechanisms underlying these contrasting functions of iNOS is unknown. Because iNOS is often induced by inflammatory signals, it is therefore likely that these contrasting functions of iNOS could be controlled by the inflammatory signaling pathways, which remains to be determined. METHODS: iNOS is expressed in colon carcinoma and myeloid cells in the tumor microenvironment. Colon carcinoma and myeloid cell lines were used to elucidate the molecular mechanisms underlying iNOS expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay were used to determine the IFNγ-activated pSTAT1 and NF-κB association with the chromatin DNA of the nos2 promoter. RESULTS: We show here that iNOS is dramatically up-regulated in inflammed human colon tissues and in human colon carcinoma as compared to normal colon tissue. iNOS is expressed in either the colon carcinoma cells or immune cells within the tumor microenvironment. On the molecular level, the proinflammatory IFNγ and NF-κB signals induce iNOS expression in human colon cancer cells. We further demonstrate that NF-κB directly binds to the NOS2 promoter to regulate iNOS expression. Although neither the IFNγ signaling pathway nor the NF-κB signaling pathway alone is sufficient to induce iNOS expression in myeloid cells, IFNγ and NF-κB synergistically induce iNOS expression in myeloid cells. Furthermore, we determine that IFNγ up-regulates IRF8 expression to augment NF-κB induction of iNOS expression. More interestingly, we observed that the p65/p65 and p50/p50 homodimers, not the canonical p65/p50 heterodimer, directly binds to the nos2 promoter to regulate iNOS expression in myeloid cells. CONCLUSIONS: IFNγ-induced IRF8 acts in concert with NF-κB to regulate iNOS expression in both colon carcinoma and myeloid cells. In myeloid cells, the NF-κB complexes that bind to the nos2 promoter are p65/p65 and p50/p50 homodimers.

Hepatic abscess associated with Salmonella serotype B in a chronic alcoholic patient.

BACKGROUND: Hepatic abscess secondary to Salmonella is extremely rare in the United States and other Western countries. METHODS: A 43-year-old Caucasian man, with a history of chronic alcohol abuse, was admitted to the hospital for intermittent abdominal pain, fever and diarrhea. Clinical, radiological and laboratory results were analyzed. Medical literature in PubMed pertaining to similar cases was reviewed. RESULTS: Stool culture was positive for Salmonella serotype B and a CT scan of the abdomen with contrast was consistent with a solitary hepatic abscess. Appropriate intravenous antibiotics followed by oral maintenance therapy for six weeks resulted in a complete clinical recovery and radiographic resolution. CONCLUSIONS: The cause of Salmonella hepatic abscess in our patient was most likely associated with decreased mucosal resistance to the bacteria, seeding of infection via transient portal bacteremia and loss of host immunity. Our case highlights the fact that appropriate antibiotic alone is sufficient in the management of a solitary hepatic abscess less than 3-5 cm.

Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.

The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic ß-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically only to those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.

TGF-beta isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas.

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.

Faulty bone morphogenetic protein signaling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: The organogenesis of esophageal atresia with tracheoesophageal fistula remains unclear. We have previously demonstrated that the fistula tract develops from a trifurcation of the embryonic lung bud and displays pulmonary lineage traits. Unlike the lung, the fistula grows without branching. Bone morphogenetic proteins (BMPs) are known to be important in lung branching. We studied possible BMP signaling defects as a potential cause for the absence of branching in the fistula tract. METHODS: Adriamycin was administered to pregnant rats on days 6-9 of gestation to induce tracheoesophageal fistula. Microdissection was performed at E13 and E17 isolating the foregut. Tissues were analyzed using immunohistochemistry for BMP ligand (BMP2, BMP4, BMP7) and receptor (BMPRIA, BMPRIB, BMPRII) expression. RESULTS: Immunohistochemistry revealed the presence of all 3 BMP ligands at E13, localized specifically to the esophageal mucosa but absent in the fistula and lung. At E17, the ligands were again present in the esophageal mucosa, and additionally in the fistula tract mucosa, but remained absent in the lung. At E17, all of the BMP receptors were also localized to the luminal surface of esophagus and fistula. However, in the lung epithelium, only BMPRII was found, whereas BMPRIA and BMPRIB remained absent. CONCLUSIONS: The normal expression pattern of BMP4 was increased at the branch tips and low between branches. Among other results, we show here a constant expression level of BMP ligands throughout the entire epithelium of the fistula tract. This diffuse expression suggests defective BMP signaling in the fistula tract and explains its nonbranching phenotype.

Mesenchymal epimorphin is important for pancreatic duct morphogenesis.

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.

Bone morphogenetic protein expression patterns in human esophageal atresia with tracheoesophageal fistula.

The organogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown. The fistula tract appears to develop from a non-branching trifurcation of the embryonic lung bud. The non-branching growth of the fistula differs from the other lung buds and suggests a deficiency in bone morphogenetic protein (BMP) signaling, since BMPs are critical to proper lung development and branching. With IRB approval, portions of newborn human proximal esophageal pouch and distal fistula samples were recovered at the time of surgical repair of EA/TEF. The tissues were processed for immunohistochemistry. Commercially available fetal tissues were used as controls. In control tissues, BMP ligands (BMP 2, 4, and 7) were all present in the esophagus but absent in the trachea. BMPRIA was absent in both tissues. BMPRIB was detected in trachea but not in esophagus and BMPRII was detected in esophagus but not in trachea. In the EA/TEF specimens, all BMP ligands were present in the proximal esophageal pouch but absent in the fistula tract. BMPRIA and BMPRIB were not detected in either tissue. However, BMPRII was found in both fistula tract and proximal pouch. The submucosa of the fistula appears to maintain a mixed (identical neither to lung, esophagus, or trachea) BMP signaling pattern, providing one mechanism which could potentially explain the esophageal dismotility and lack of lung branching seen in the fistula/distal esophagus.

Synergistic endocrine induction by GLP-1 and TGF-beta in the developing pancreas.

OBJECTIVES: Glucagon-like peptide-1 (GLP-1) is known to stimulate glucose-dependent insulin production and secretion by pancreatic beta-cells. Preliminary evidence suggests that GLP-1 may also influence endocrine differentiation from pancreatic progenitor cells. Additionally, TGF-beta signaling can also control endocrine differentiation by both inhibiting proliferation and enhancing differentiation of endocrine progenitor cells to become mature beta-cells. Here we document synergy of these two signaling pathways in the differentiation of endocrine cells in the developing pancreas. METHODS: Embryonic pancreas was harvested from mice at day 11.5 and cultured for six days with GLP-1 agonist, exendin-4, and/or TGF-beta1 ligand. Also, a pan-neutralizing TGF-beta isoform antibody was used alone or with exendin-4 to study TGF-beta inhibition in this system. Pancreatic cultures were processed for immunohistochemistry. RESULTS: Exogenous TGF-beta1 and exendin-4 each individually enhanced both insulin and glucagon differentiation dose-dependently. However, when combined there was an additive effect to a 4.5-fold increase in insulin-positive differentiation. We also saw suppression of amylase-positive differentiation. Surprisingly, TGF-beta pan-neutralizing antibody also gave an augmentation of endocrine differentiation by 1.5 to 2-fold, but no synergistic effect was seen with exendin-4. CONCLUSION: We conclude that TGF-beta isoforms have a specific synergistic role with GLP-1 pathway signaling in early pancreatic development, toward endocrine differentiation and away from acinar differentiation.

Interplay of glucagon-like peptide-1 and transforming growth factor-beta signaling in insulin-positive differentiation of AR42J cells.

The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo beta-cell differentiation as well as potential application to beta-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4-induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-beta isoform release by the AR42J cells and may serve as a mechanism to promote beta-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-beta1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-beta isoform signaling.

Hepatic regeneration and enforced PDX-1 expression accelerate transdifferentiation in liver.

BACKGROUND: Pancreatic duodenal homeobox gene-1 (PDX-1) has a dual task as a key regulator in pancreatic organogenesis and in functional maintenance of beta cells in adults. Recent studies have shown a close lineage relationship between the liver and the pancreas. In this study, we analyzed the plasticity of the liver by enforced expression of PDX-1 in streptozotocin (STZ)-treated mice under the condition of hepatic regeneration. METHODS: Replication-deficient adenoviruses were constructed by the cosmid-adenoviral DNA terminal protein complex method. Mice were treated with STZ (200 mg/kg ip), and a 40% partial hepatectomy was performed at day 0. After 24 hours, Ad-pdx-1 or Ad-lacZ 2.0 x 10(9) PFU/body was injected via the tail vain into nontreated (control), STZ-treated, or STZ plus partial hepatectomy (Hx)-treated ICR mice. After 7 and 14 days, expression of PDX-1 and islet hormones was examined by immunohistologic and reverse transcription-polymerase chain reaction analysis. Blood glucose concentrations were measured every 2 days. Immunoreactive insulin (IRI) of serum and liver extract was measured by ELISA. RESULTS: Most hepatocytes of Ad-pdx-1-infected mice were positive for PDX-1 expression by immunohistochemistry. In nontreated mice, very few cells expressed insulin and other hormones. In contrast, insulin and somatostatin were expressed in STZ-treated mice, and more cells were expressed in STZ plus Hx-treated mice. In addition, other beta-cell markers like GLUT2 and glucokinase were observed. Hyperglycemia was improved in STZ-treated mice and STZ plus Hx-treated mice. IRI of serum and liver extract was increased in STZ-treated mice and STZ plus Hx-treated mice. The insulin positive area of the liver in STZ plus Hx-treated mice was larger than that in nontreated and STZ-treated mice. CONCLUSIONS: Ectopic PDX-1 expression alone may be insufficient to induce insulin-producing cells in the liver. STZ-induced hyperglycemia plus partial hepatectomy that leads to diabetic state and hepatic regeneration may stimulate the transdifferentiation of liver cells into insulin-producing cells.

N-cadherin expression and epithelial-mesenchymal transition in pancreatic carcinoma.

PURPOSE: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors. EXPERIMENTAL DESIGN: We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)beta, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines. RESULTS: N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change. CONCLUSION: This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

Increased PDX-1 expression is associated with outcome in patients with pancreatic cancer.

BACKGROUND: During pancreatic development, pancreatic duodenal homeobox gene-1 (PDX-1) is expressed in pancreatic duct cells that have the potential to differentiate into islets. Therefore, PDX-1 is thought to be a marker of de-differentiated cells with the capacity to redifferentiate into several pancreatic cell types. We analyzed PDX-1 expression in human pancreatic cancer specimens, pancreatic cancer cell lines, and the effects of forced expression of PDX-1 in pancreatic cancer cells. METHODS: Thirty-five pancreatic adenocarcinomas were immunohistochemically stained with a polyclonal rabbit antibody against mouse PDX-1. Correlations with tumor characteristics were made with chi-squared analysis. The influence of clinicopathologic factors on survival was assessed. The expression of PDX-1 in pancreatic cancer cells was examined. Replication-deficient recombinant adenoviruses were constructed by the cosmid-adenoviral DNA terminal protein complex method. PANC-1 cells were infected with Ad-pdx-1 or Ad-LacZ. PANC-1 cells that were infected with adenovirus were used in a cell growth assay and a migration assay and for morphologic analysis. RESULTS: Interestingly, 43% of pancreatic cancers were positive for PDX-1 expression, and 57% of pancreatic cancers were negative (normal pancreatic exocrine tissue shows little or no staining for PDX-1). Lymph node metastasis (P =.02) and histologic grade (P =.04) were correlated significantly with PDX-1 expression. Patients with positive PDX-1 had a significantly worse prognosis than those patients with negative PDX-1 (P =.02). Importantly, PDX-1 was an independent variable that effected overall survival (P =.03). Pancreatic cancer cell lines showed no PDX-1 expression. There were no significant differences in cell proliferation or morphologic condition between Ad-pdx-1- and Ad-lacZ-infected PANC-1 cells. However, Ad-pdx-1-infected PANC-1 cells did show a significantly higher migration rate than Ad-lacZ-infected PANC-1 cells. CONCLUSIONS: Re-expression of PDX-1 may represent a return to a more de-differentiated state by more aggressive pancreatic cancers and may also represent an important new tumor marker for these aggressive cancers.

Endogenous decoy receptor 3 blocks the growth inhibition signals mediated by Fas ligand in human pancreatic adenocarcinoma.

Many cancers are resistant to Fas-mediated apoptosis despite the expression of Fas. To investigate the mechanisms by which Fas signals are attenuated, we focused on decoy receptor 3 (DcR3). DcR3 is a soluble receptor against Fas ligand belonging to the tumor necrosis factor receptor superfamily and overexpresses in some forms of cancers. Exogenous DcR3 inhibits Fas-mediated apoptosis in Fas-sensitive Jurkat cells. In our study, we examined the expression and function of DcR3 in pancreatic cancers. TaqMan RT-PCR showed that DcR3 mRNA was highly expressed in pancreatic cancer cell lines (71%) and tissues (67%). Its expression significantly correlated with cancer invasion to veins. Western blotting showed that the DcR3 protein was produced and secreted in 4 of 6 cell lines. The protein expressions were compatible with the mRNA expression. Five of 7 pancreatic cancer cell lines became sensitive to agonistic anti-Fas antibody (CH-11) to various extents, without Fas upregulation, when exposed to CH-11 for 48 hr after pretreatment with IFNgamma. Four of 7 pancreatic cancer cell lines were inhibited from growing, compared to control cells, when cocultured with membrane-bounded Fas ligand (mFasL) transfected lymphomas for 48 hr after pretreatment with IFNgamma. DcR3 reduced this growth inhibition when added exogenously. Regression analysis showed that the DcR3 expression significantly correlated with the sensitivity to mFasL, and not to CH-11. These results suggest that DcR3 is highly expressed in many pancreatic cancers and endogenous DcR3 blocks the growth inhibition signals mediated by mFasL. DcR3 can be a candidate target molecule for the therapeutic intervention.