Hydrogen peroxide is a diffusible paracrine signal for the induction of epithelial cell death by activated myofibroblasts

Cell‐cell signaling roles for reactive oxygen species (ROS) generated in response to growth factors/cytokines in nonphagocytic cells are not well defined. In this study, we show that fibroblasts isolated from lungs of patients with idiopathic pulmonary fibrosis (IPF) generate extracellular hydrogen peroxide (H2O2) in response to the multifunctional cytokine, transforming growth factor‐β1 (TGF‐β1). In contrast, TGF‐β1 stimulation of small airway epithelial cells (SAECs) does not result in detectable levels of extracellular H2O2. IPF fibroblasts independently stimulated with TGF‐β1 induce loss of viability and death of overlying SAECs when cocultured in a compartmentalized Transwell system. These effects on SAECs are inhibited by the addition of catalase to the coculture system or by the selective enzymatic blockade of H2O2 production by IPF fibroblasts. IPF fibroblasts heterogeneously express α‐smooth muscle actin stress fibers, a marker of myofibroblast differentiation. Cellular localization of H2O2 by a fluorescent‐labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast phenotype. Thus, myofibroblast secretion of H2O2 functions as a diffusible death signal for lung epithelial cells. This novel mechanism for intercellular ROS signaling may be important in physiological/pathophysiological processes characterized by regenerating epithelial cells and activated myofibroblasts.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154383/1/fsb2fj042882fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154383/2/fsb2fj042882fje-sup-0001.pd

Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for >10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival <14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFβ-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that 'High-RPS8 and Low-PRELP' was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46-4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated

Cell Specific Regulation of Sonic Hedgehog in Adult Stomach: Understanding Mechanisms Leading to Gastric Atrophy/Metaplasia. Mechanisms Leading to Gastric Atrophy/Metaplasia.

Helicobacter induced gastritis of the corpus results in loss of parietal cell function, loss of the oxyntic gland (atrophy), changes that predispose to gastric cancer. The molecular details of how chronic inflammation in the corpus leads to atrophy is not well understood. Recently, Shh has been implicated as a crucial factor in gastric gland organogenesis and differentiation. Several studies have shown in both human subjects and rodent models that Helicobacter infection leads to a decrease in Shh expression. However, none of the studies to date have examined a role for specific pro-inflammatory cytokines in the regulation of Shh expression. Polymorphisms in the promoter of the pro-inflammatory cytokine IL-1betathat increase gene expression correlate with gastric atrophy and cancer in Helicobacter-infected subjects. Recently, gastric transgenic overexpression of human IL-1beta in mice was shown to induce dysplasia and gastric cancer. Apart from its role in inflammation, IL-1beta inhibits gastric acid secretion and hence in this way might hasten atrophy. Prior studies have suggested a role for gastric acid in regulating Shh expression. Therefore, I tested whether IL-1beta induces gastric atrophy through its ability to inhibit Shh gene expression. There is conflicting information about the cell types that express Shh in the stomach. Therefore using reporter mice, I first determined the patterns of Shh expression. I found that all major cell lineages of the corpus express Shh and that Hh signaling in the stomach is paracrine. Next, I investigated the ability of Helicobacter-induced inflammation to regulate Shh gene expression by infecting ShhLacZ reporter mice. Helicobacter infection inhibited Shh expression in parietal cells. In addition I found that IL-1 also inhibited Shh expression in the parietal cells. Activation of the IL-1receptor was required to inhibit Shh expression by IL-1beta. As previously reported, I found that IL-1beta inhibited acid secretion. Further I demonstrated that IL-1beta inhibits Shh gene expression by both acid dependent and independent mechanisms. In summary, I have established a previously unknown role for IL-1beta in gastric pathophysiology, that is, its ability to suppress Shh gene expression, a factor essential to the functional maturity of the parietal cell.Ph.D.Cell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62276/1/mwaghray_1.pd

Histamine 3 receptor activation mediates inhibition of acid secretion during Helicobacter-induced gastritis

AIM: To test the hypothesis that histamine 3 receptor (H3R) activation during Helicobacter infection inhibits gastric acid secretion in vivo and in vitro

Bmi1 enhances tumorigenicity and cancer stem cell function in pancreatic adenocarcinoma.

Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma.We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing.Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue.Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells

Inflammation and Gli2 Suppress Gastrin Gene Expression in a Murine Model of Antral Hyperplasia

<div><p>Chronic inflammation in the stomach can lead to gastric cancer. We previously reported that gastrin-deficient (<em>Gast^−/−</em>) mice develop bacterial overgrowth, inflammatory infiltrate, increased Il-1β expression, antral hyperplasia and eventually antral tumors. Since Hedgehog (Hh) signaling is active in gastric cancers but its role in precursor lesions is poorly understood, we examined the role of inflammation and Hh signaling in antral hyperplasia. <em>LacZ</em> reporter mice for <em>Sonic hedgehog</em> (<em>Shh</em>), <em>Gli1</em>, and <em>Gli2</em> expression bred onto the <em>Gast^−/−</em> background revealed reduced <em>Shh</em> and <em>Gli1</em> expression in the antra compared to wild type controls (WT). <em>Gli2</em> expression in the <em>Gast^−/−</em> corpus was unchanged. However in the hyperplastic <em>Gast^−/−</em> antra, <em>Gli2</em> expression increased in both the mesenchyme and epithelium, whereas expression in WT mice remained exclusively mesenchymal. These observations suggested that <em>Gli2</em> is differentially regulated in the hyperplastic <em>Gast^−/−</em> antrum versus the corpus and by a Shh ligand-independent mechanism. Moreover, the proinflammatory cytokines Il-1β and Il-11, which promote gastric epithelial proliferation, were increased in the <em>Gast^−/−</em> stomach along with Infγ. To test if inflammation could account for elevated epithelial <em>Gli2</em> expression in the <em>Gast^−/−</em> antra, the human gastric cell line AGS was treated with IL-1β and was found to increase <em>GLI2</em> but decrease <em>GLI1</em> levels. IL-1β also repressed human <em>GAST</em> gene expression. Indeed, GLI2 but not GLI1 or GLI3 expression repressed gastrin luciferase reporter activity by ∼50 percent. Moreover, chromatin immunoprecipitation of GLI2 in AGS cells confirmed that GLI2 directly binds to the <em>GAST</em> promoter. Using a mouse model of constitutively active epithelial GLI2 expression, we found that activated GLI2 repressed <em>Gast</em> expression but induced <em>Il-1β</em> gene expression and proliferation in the gastric antrum, along with a reduction of the number of G-cells. In summary, epithelial Gli2 expression was sufficient to stimulate <em>Il-1β</em> expression, repress <em>Gast</em> gene expression and increase proliferation, leading to antral hyperplasia.</p> </div

Modulation of Bmi1 expression affects tumor growth and invasion <i>in vivo</i>.

<p><b>A</b>. Representative images shown demonstrate relative tumor size difference at 28 days following orthotopic pancreas implantation of MiaPaCa2 (top) and Panc-1 cells (bottom) in NOD-SCID mice after modulating Bmi1 expression. The tumor volume was calculated by using the formula for volume of an ellipsoid structure (4/3 * π * width/2 * length/2 * height/2). Data are expressed as the mean ± SEM (* p<0.05, n = 5 independent experiments). <b>B</b>. The image shown is representative of enhanced metastatic potential in MiaPaCa2 cells following induced expression of Bmi1. * Marked sites show gross metastasis of MiaPaCa2 tumor to extrapancreatic sites in NOD-SCID mice 28 days following orthotopic injection of cells.</p