Indication for differential sorting of the rat v-SNARE splice isoforms VAMP-1a and -1b.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential constituents of the intracellular trafficking machinery. The variable C-terminus in the 2 rat VAMP-1 splice isoforms VAMP-1a and -1b potentially acts as a sorting signal, because similar changes at the C-terminal end of a human VAMP-1 splice isoform resulted in its sorting to mitochondria. To evaluate the differences in the subcellular localization of these two v-SNARE proteins, VAMP-1a and -1b proteins tagged with green fluorescent protein (GFP) and red fluorescent protein (RFP) were expressed in HeLa, COS-7, and MDCK cells and evaluated by conventional confocal as well as total internal reflection fluorescence microscopy. Regions consistent with the endoplasmic reticulum and Golgi apparatus demonstrated a major overlap of both signals. In the periphery, vesicular structures were observed that mainly expressed one of the 2 isoforms. Within our experimental settings, we could not observe sorting of any of the 2 isoforms to mitochondria or peroxisomes, whereas both isoforms were found expressed in a minor subset of singular vesicles, which sporadically appeared to co-localize with the exocyst marker EXOC3/Sec6. Because vesicular structures were seen that expressed only one of the two splice variants, it is possible that VAMP-1a and VAMP-1b are sorted to distinct cellular compartments that require further characterization.

Epidermal growth factor receptor (EGFR) signaling requires a specific endoplasmic reticulum thioredoxin for the post-translational control of receptor presentation to the cell surface.

The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.

Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Loss of anterior gradient 2 (Agr2) expression results in hyperplasia and defective lineage maturation in the murine stomach.

Recent studies of epithelial tissues have revealed the presence of tissue-specific stem cells that are able to establish multiple cell lineages within an organ. The stem cells give rise to progenitors that replicate before differentiating into specific cell lineages. The mechanism by which homeostasis is established between proliferating stem or progenitor cells and terminally differentiated cells is unclear. This study demonstrates that Agr2 expression by mucous neck cells in the stomach promotes the differentiation of multiple cell lineages while also inhibiting the proliferation of stem or progenitor cells. When Agr2 expression is absent, gastric mucous neck cells increased in number as does the number of proliferating cells. Agr2 expression loss also resulted in the decline of terminally differentiated cells, which was supplanted by cells that exhibited nuclear SOX9 labeling. Sox9 expression has been associated with progenitor and stem cells. Similar effects of the Agr2 null on cell proliferation in the intestine were also observed. Agr2 consequently serves to maintain the balance between proliferating and differentiated epithelial cells.

Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1.

BACKGROUND & AIMS: Early detection of pancreatic ductal adenocarcinoma (PDAC) allows for surgical resection and increases patient survival times. Imaging agents that bind and amplify the signal of neovascular proteins in neoplasms can be detected by ultrasound, enabling accurate detection of small lesions. We searched for new markers of neovasculature in PDAC and assessed their potential for tumor detection by ultrasound molecular imaging. METHODS: Thymocyte differentiation antigen 1 (Thy1) was identified as a specific biomarker of PDAC neovasculature by proteomic analysis. Up-regulation in PDAC was validated by immunohistochemical analysis of pancreatic tissue samples from 28 healthy individuals, 15 with primary chronic pancreatitis tissues, and 196 with PDAC. Binding of Thy1-targeted contrast microbubbles was assessed in cultured cells, in mice with orthotopic PDAC xenograft tumors expressing human Thy1 on the neovasculature, and on the neovasculature of a genetic mouse model of PDAC. RESULTS: Based on immunohistochemical analyses, levels of Thy1 were significantly higher in the vascular of human PDAC than chronic pancreatitis (P = .007) or normal tissue samples (P < .0001). In mice, ultrasound imaging accurately detected human Thy1-positive PDAC xenografts, as well as PDACs that express endogenous Thy1 in genetic mouse models of PDAC. CONCLUSIONS: We have identified and validated Thy1 as a marker of PDAC that can be detected by ultrasound molecular imaging in mice. The development of a specific imaging agent and identification of Thy1 as a new biomarker could aid in the diagnosis of this cancer and management of patients.

AGR2 gene function requires a unique endoplasmic reticulum localization motif.

Soluble proteins are enriched in the endoplasmic reticulum (ER) by retrograde transport from the Golgi that is mediated by the KDEL receptors. In addition to the classic carboxyl-terminal KDEL motif, a variety of sequence variants are also capable of receptor binding that result in ER localization. Although different ER localization signals that exhibit varying affinities for the KDEL receptors exist, whether there are functional implications was unknown. The present study determines whether AGR2 requires a specific ER localization signal to be functionally active. AGR2 is expressed in most human adenocarcinomas and serves a role in promoting growth and the transformed phenotype. Using two different cell lines in which AGR2 induces expression of either the EGFR ligand amphiregulin or the transcription factor CDX2, only the highly conserved wild-type carboxyl-terminal KTEL motif results in the appropriate outcome. Deletion of the KTEL motif results in AGR2 secretion and loss of AGR2 function. AGR2 function is also lost when ER residence is achieved with a carboxyl-terminal KDEL or KSEL instead of a KTEL motif. Thus variations in ER localization sequences may serve a specific functional role, and in the case of AGR2, this role is served specifically by KTEL.

CITED2 is a novel direct effector of peroxisome proliferator-activated receptor γ in suppressing hepatocellular carcinoma cell growth.

BACKGROUND: Previous reports from these authors found that activation of peroxisome proliferator-activated receptor gamma (PPARγ) suppressed hepatocellular carcinoma (HCC). This study sought to identify the molecular target of PPARγ and characterize its antitumor effect in HCC. METHODS: Optimal PPARγ binding activity was obtained using the PPARγ agonist rosiglitazone (100 µM) as determined by enzyme-linked immunosorbent assay. Under PPARγ activation, 114 PPARγ downstream targets associated with cancer development were identified by oligonucleotide microarray and Gene Ontology analysis. Among them, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) was the most prominent PPARγ-bound target, as determined by chromatin immunoprecipitation-polymerase chain reaction. RESULTS: CITED2 messenger RNA and protein was significantly down-regulated in primary HCCs compared with their adjacent nontumor tissues. PPARγ induced expression of CITED2 in HCC cell lines after adenovirus-PPARγ transduction. The biological function of CITED2 was evaluated by loss- and gain-of-function assays. CITED2 knockdown in the hepatocyte cell line LO2 and HCC cell line Hep3B significantly increased cell viability and clonogenicity, and promoted G1 -S phase transition in both cell lines. In contrast, ectopic expression of CITED2 in HepG2 and BEL7404 HCC cell lines significantly suppressed cell growth. The tumor suppressive effect of CITED2 was associated with up-regulation of cyclin-dependent kinase inhibitors p15(INK4B) , p21(Wat1/Cip1) , p27(Kip1) , antiproliferative regulator interferon alpha 1, proapoptotic mediators including tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), TNFRSF25, caspase-8, granzyme A, and the tumor suppressor gene maspin. CITED2 was also associated with the down-regulation of cell cycle regulator cyclin D1, oncogene telomerase reverse transcriptase, and proinvasion/metastasis gene matrix metallopeptidase 2. CONCLUSIONS: CITED2 is a direct effector of PPARγ for tumor suppression. Cancer 2013. © 2012 American Cancer Society.

Metabolomic-derived novel cyst fluid biomarkers for pancreatic cysts: glucose and kynurenine.

BACKGROUND: Better pancreatic cyst fluid biomarkers are needed. OBJECTIVE: To determine whether metabolomic profiling of pancreatic cyst fluid would yield clinically useful cyst fluid biomarkers. DESIGN: Retrospective study. SETTING: Tertiary-care referral center. PATIENTS: Two independent cohorts of patients (n = 26 and n = 19) with histologically defined pancreatic cysts. INTERVENTION: Exploratory analysis for differentially expressed metabolites between (1) nonmucinous and mucinous cysts and (2) malignant and premalignant cysts was performed in the first cohort. With the second cohort, a validation analysis of promising identified metabolites was performed. MAIN OUTCOME MEASUREMENTS: Identification of differentially expressed metabolites between clinically relevant cyst categories and their diagnostic performance (receiver operating characteristic [ROC] curve). RESULTS: Two metabolites had diagnostic significance-glucose and kynurenine. Metabolomic abundances for both were significantly lower in mucinous cysts compared with nonmucinous cysts in both cohorts (glucose first cohort P = .002, validation P = .006; and kynurenine first cohort P = .002, validation P = .002). The ROC curve for glucose was 0.92 (95% confidence interval [CI], 0.81-1.00) and 0.88 (95% CI, 0.72-1.00) in the first and validation cohorts, respectively. The ROC for kynurenine was 0.94 (95% CI, 0.81-1.00) and 0.92 (95% CI, 0.76-1.00) in the first and validation cohorts, respectively. Neither could differentiate premalignant from malignant cysts. Glucose and kynurenine levels were significantly elevated for serous cystadenomas in both cohorts. LIMITATIONS: Small sample sizes. CONCLUSION: Metabolomic profiling identified glucose and kynurenine to have potential clinical utility for differentiating mucinous from nonmucinous pancreatic cysts. These markers also may diagnose serous cystadenomas.

The human adenocarcinoma-associated gene, AGR2, induces expression of amphiregulin through Hippo pathway co-activator YAP1 activation.

Anterior Gradient Homolog 2 (AGR2) is expressed by the normal intestine and by most human adenocarcinomas, including those derived from the esophagus, pancreas, lung, breast, ovary, and prostate. Xenografts of human adenocarcinoma cell lines in nude mice previously demonstrated that AGR2 supports tumor growth. In addition, AGR2 is able to induce in vitro a transformed phenotype in fibroblast and epithelial cell lines. The mechanism underlying the growth promoting effects of AGR2 is unknown. The present study shows that AGR2 induces expression of amphiregulin (AREG), a growth promoting EGFR ligand. Induced AREG expression in adenocarcinoma cells is able to rescue the transformed phenotype that is lost when AGR2 expression is reduced. Additional experiments demonstrate that AGR2 induction of AREG is mediated by activation of the Hippo signaling pathway co-activator, YAP1. Thus AGR2 promotes growth by regulating the Hippo and EGF receptor signaling pathways.

PDX1 regulation of FABP1 and novel target genes in human intestinal epithelial Caco-2 cells.

The transcription factor pancreatic and duodenal homeobox 1 (PDX1) plays an essential role in pancreatic development and in maintaining proper islet function via target gene regulation. Few intestinal PDX1 targets, however, have been described. We sought to define novel PDX1-regulated intestinal genes. Caco-2 human intestinal epithelial cells were engineered to overexpress PDX1 and gene expression profiles relative to control cells were assessed. Expression of 80 genes significantly increased while that of 49 genes significantly decreased more than 4-fold following PDX1 overexpression in differentiated Caco-2 cells. Analysis of the differentially regulated genes with known functional annotations revealed genes encoding transcription factors, growth factors, kinases, digestive glycosidases, nutrient transporters, nutrient binding proteins, and structural components. The gene for fatty acid binding protein 1, liver, FABP1, is repressed by PDX1 in Caco-2 cells. PDX1 overexpression in Caco-2 cells also results in repression of promoter activity driven by the 0.6kb FABP1 promoter. PDX1 regulation of promoter activity is consistent with the decrease in FABP1 RNA abundance resulting from PDX1 overexpression and identifies FABP1 as a candidate PDX1 target. PDX1 repression of FABP1, LCT, and SI suggests a role for PDX1 in patterning anterior intestinal development.

Diagnostic accuracy of cyst fluid amphiregulin in pancreatic cysts.

BACKGROUND: Accurate tests to diagnose adenocarcinoma and high-grade dysplasia among mucinous pancreatic cysts are clinically needed. This study evaluated the diagnostic utility of amphiregulin (AREG) as a pancreatic cyst fluid biomarker to differentiate non-mucinous, benign mucinous, and malignant mucinous cysts. METHODS: A single-center retrospective study to evaluate AREG levels in pancreatic cyst fluid by ELISA from 33 patients with a histological gold standard was performed. RESULTS: Among the cyst fluid samples, the median (IQR) AREG levels for non-mucinous (n = 6), benign mucinous (n = 15), and cancerous cysts (n = 15) were 85 pg/ml (47-168), 63 pg/ml (30-847), and 986 pg/ml (417-3160), respectively. A significant difference between benign mucinous and malignant mucinous cysts was observed (p = 0.025). AREG levels greater than 300 pg/ml possessed a diagnostic accuracy for cancer or high-grade dysplasia of 78% (sensitivity 83%, specificity 73%). CONCLUSION: Cyst fluid AREG levels are significantly higher in cancerous and high-grade dysplastic cysts compared to benign mucinous cysts. Thus AREG exhibits potential clinical utility in the evaluation of pancreatic cysts.

The pancreatic stellate cell: a star on the rise in pancreatic diseases.

Pancreatic stellate cells (PaSCs) are myofibroblast-like cells found in the areas of the pancreas that have exocrine function. PaSCs are regulated by autocrine and paracrine stimuli and share many features with their hepatic counterparts, studies of which have helped further our understanding of PaSC biology. Activation of PaSCs induces them to proliferate, to migrate to sites of tissue damage, to contract and possibly phagocytose, and to synthesize ECM components to promote tissue repair. Sustained activation of PaSCs has an increasingly appreciated role in the fibrosis that is associated with chronic pancreatitis and with pancreatic cancer. Therefore, understanding the biology of PaSCs offers potential therapeutic targets for the treatment and prevention of these diseases.