Hyperproliferation Of Epithelial Cells Research Articles

Expression of Circadian Clock Components PER2 and BMAL1 are Altered During Infection of Helicobacter pylori

Background/HypothesisSeveral gastrointestinal functions have been demonstrated to be under circadian clock control and colorectal cancer tissues have been found to have a disruption in core circadian clock components BMAL1 and PER2. Those suffering from chronic Helicobacter pylori (H. pylori) infection are at a heightened risk of developing gastric adenocarcinoma. Compared to young individuals, the aged population suffer from an increased susceptibility to H. pylori infection as well as diminished expression of circadian clock components which could further predispose them to the development of gastric cancer. We therefore hypothesize that chronic H. pylori infection induces a hyperproliferative response in the gastric epithelium via BMAL1 activation resulting in a susceptibility to gastric cancer.MethodsGastric organoids (hFGOs) derived from primary gastric tissue of either aged (> 55 years of age) or young (14 – 20 years) individuals were generated and subsequently infected with H. pylori. Analysis for proliferation and expression of circadian clock components BMAL1 and PER2 was performed for a period of 24 hours post‐infection. Organoids infected with H. pylori were analyzed for gastric cancer markers by qRT‐PCR.ResultsGastric fundic tissue obtained from individuals infected with H. pylori were found to have a significantly upregulated expression of cytoplasmic BMAL1. hFGOs exhibited a robust circadian expression of Bmal1, Per2, and phosphorylated (inactive) GSK‐3β. In comparison to uninfected controls, hFGOs infected with H. pylori displayed a heightened expression of BMAL1, phosphorylated GSK‐3β, and epithelial proliferation. Gastric organoids derived from of elderly patients was found to have a diminished rhythmic expression of clock genes Per2 and Bmal1. This dampening in expression of circadian clock components correlates with an increased expression of markers for intestinal metaplasia.ConclusionsPersistent infection by H. pylori inactivates GSK‐3β resulting in the activation of the BMAL1:CLOCK complex leading to a hyperproliferation in gastric epithelial cells. This mechanism could heighten the risk of developing gastric cancer in the aged population and in individuals with chronic H. pylori infection. Per2 knockout mice have been generated and infected with H. pylori in order to further understand this mechanism in vivo. Results are ongoing and pending.Support or Funding InformationNIH 1U19AI116491‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

β-catenin ablation exacerbates polycystic kidney disease progression.

Polycystic kidney disease (PKD) results from excessive renal epithelial cell proliferation, leading to the formation of large fluid filled cysts which impair renal function and frequently lead to renal failure. Hyperactivation of numerous signaling pathways is hypothesized to promote renal epithelial cell hyperproliferation including mTORC1, extracellular signal-regulated kinase (ERK) and WNT signaling. β-catenin and its target genes are overexpressed in some PKD models and expression of activated β-catenin induces cysts in mice; however, β-catenin murine knockout studies indicate it may also inhibit cystogenesis. Therefore, it remains unclear whether β-catenin is pro- or anti-cystogenic and whether its role is canonical WNT signaling-dependent. Here, we investigate whether β-catenin deletion in a PKD model with hyperactived β-catenin signaling affects disease progression to address whether increased β-catenin drives PKD. We used renal epithelial cell specific Inpp5e-null PKD mice which we report exhibit increased β-catenin and target gene expression in the cystic kidneys. Surprisingly, co-deletion of β-catenin with Inpp5e in renal epithelial cells exacerbated polycystic kidney disease and renal failure compared to Inpp5e deletion alone, but did not normalize β-catenin target gene expression. β-catenin/Inpp5e double-knockout kidneys exhibited increased cyst initiation, cell proliferation and MEK/ERK signaling compared to Inpp5e-null, associated with increased fibrosis, which may collectively contribute to accelerated disease. Therefore, increased β-catenin and WNT target gene expression are not necessarily cyst promoting. Rather β-catenin may play a dual and context-dependent role in PKD and in the presence of other cyst-inducing mutations (Inpp5e-deletion); β-catenin loss may exacerbate disease in a WNT target gene-independent manner.

Lycopene treatment inhibits activation of Jak1/Stat3 and Wnt/β-catenin signaling and attenuates hyperproliferation in gastric epithelial cells

Helicobacter pylori (H pylori) colonizes the human stomach and increases the risk of gastric diseases including gastric cancer. H pylori increases reactive oxygen species (ROS), which activate Janus-activator kinase 1 (Jak1)/signal transducers and activators of transcription 3 (Stat3) in gastric epithelial cells. ROS mediate hyperproliferation, a hallmark of carcinogenesis, by activating Wnt/β-catenin signaling in various cells. Lycopene is a potent antioxidant exhibiting anticancer effects. We hypothesized that lycopene may inhibit H pylori–induced hyperproliferation by suppressing ROS-mediated activation of Jak1/Stat3 and Wnt/β-catenin signaling, and β-catenin target gene expression in gastric epithelial cells. We determined cell viability, ROS levels, and the protein levels of phospho- and total Jak1/Stat3, Wnt/β-catenin signaling molecules, Wnt-1, lipoprotein-related protein 5, and β-catenin target oncogenes (c-Myc and cyclin E) in H pylori–infected gastric epithelial AGS cells. The Jak1/Stat3 inhibitor AG490 served as the control treatment. The significance of the differences among groups was calculated using the 1-way analysis of variance followed by Newman-Keuls post hoc tests. The results show that lycopene reduced ROS levels and inhibited Jak1/Stat3 activation, alteration of Wnt/β-catenin multiprotein complex molecules, expression of c-Myc and cyclin E, and cell proliferation in H pylori–infected AGS cells. AG490 similarly inhibited H pylori–induced cell proliferation, alteration of Wnt/β-catenin multiprotein complex molecules, and oncogene expression. H pylori increased the levels of Wnt-1 and its receptor lipoprotein-related protein 5; this increase was inhibited by either lycopene or AG490 in AGS cells. In conclusion, lycopene inhibits ROS-mediated activation of Jak1/Stat3 and Wnt/β-catenin signaling and, thus, oncogene expression in relation to hyperproliferation in H pylori–infected gastric epithelial cells. Lycopene might be a potential and promising nutrient for preventing H pylori–associated gastric diseases including gastric cancer.

Chronic ingestion of deoxynivalenol at human dietary levels impairs intestinal homeostasis and gut microbiota in mice

The mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereals and their by-products in areas with a moderate climate. Produced by Fusarium species, it is one of the most prevalent mycotoxins in cereal crops worldwide, and the most frequently occurring type B trichothecene in Europe. Due to its toxic properties, high stability and prevalence, the presence of DON in the food chain could represent a major public health risk. However, despite its well-known acute toxicological effects, information on the adverse effects of realistic exposure remains limited. We orally exposed mice during 9months to DON at doses relevant for currently estimated human intake and explored the impact on various gut health parameters. DON exposure induced recruitment of regulatory B cells, and activation of regulatory T cells and dendritic cells in mesenteric lymph nodes. Several inflammatory parameters were increased in colon of DON-exposed mice, whereas inversely inflammatory markers were decreased in ileum. Histomorphological impairments were observed from the duodenum to the colon. Both colon and jejunum presented a hyperproliferation of epithelial cells and an increased expression of mature absorptive cells markers. Finally, DON exposure reshaped gut microbial structure and drastically disturbed the abundance of several bacterial phyla, families, and genera, leading to dysbiosis. Chronic oral exposure to human relevant doses of DON induces several disturbances of gut homeostasis with likely pathological implications for susceptible individuals.

P-167 - Astaxanthin inhibits Helicobacter pylori-induced hyper-proliferation in gastric epithelial cells

Helicobacter pylori (H. pylori) causes peptic ulcer disease, gastritis and gastric adenocarcinoma in gastric epithelial cells. Hyper-proliferation is observed in the mucosa of H. pylori-infected patients. H. pylori stimulates production of reactive oxygen species (ROS), which mediates hyper-proliferation of the infected gastric epithelial cells. Astaxanthin (AST) is a natural carotenoid showing a powerful anti-oxidative effect. The present study was purposed to investigate whether AST inhibits H. pylori-induced hyper-proliferation in gastric epithelial cells. As a result, H. pylori induced hyper-proliferation and increased levels of intracellular ROS in gastric epithelial AGS cells. In addition, H. pylori increased mitochondrial ROS levels by disturbing mitochondrial function, determined by mitochondrial membrane potential. AST inhibited H. pylori-induced hyper-proliferation and increases in intracellular and mitochondrial ROS levels in AGS cells. In conclusion, supplementation of AST may be beneficial for preventing H. pylori-associated gastric disorders by reducing ROS levels and hyper-proliferation in H. pylori-infected gastric epithelial cells.

Nrf2 regulates cellular behaviors and Notch signaling in oral squamous cell carcinoma cells

Oxidative stress is known to play a pivotal role in the development of oral squamous cell carcinoma (OSCC). We have demonstrated that activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway has chemopreventive effects against oxidative stress-associated OSCC. However, Nrf2 have dual roles in cancer development; while it prevents carcinogenesis of normal cells, hyperactive Nrf2 also promotes the survival of cancer cells. This study is aimed to understand the function of Nrf2 in regulating cellular behaviors of OSCC cells, and the potential mechanisms through which Nrf2 facilitates OSCC. We established the Nrf2-overexpressing and Nrf2-knockdown OSCC cell lines, and examined the function of Nrf2 in regulating cell proliferation, migration, invasion, cell cycle and colony formation. Our data showed that Nrf2 overexpression promoted cancer phenotypes in OSCC cells, whereas Nrf2 silencing inhibited these phenotypes. In addition, Nrf2 positively regulated Notch signaling pathway in OSCC cells in vitro. Consistent with this observation, Nrf2 activation in Keap1−/− mice resulted in not only hyperproliferation of squamous epithelial cells in mouse tongue as evidenced by increased expression of PCNA, but also activation of Notch signaling in these cells as evidenced by increased expression of NICD1 and Hes1. In conclusion, Nrf2 regulates cancer behaviors and Notch signaling in OSCC cells.

Abstract 1417: Expression of BARD1δ induces genetic instability and is associated with cervical carcinogenesis

Abstract Cervical cancer is always linked to infection with the human papilloma virus (HPV), which causes hyper-proliferation of cervical epithelial cells and neoplasia and cervical cancer. The viral oncoprotein interaction with host tumor suppressor proteins partially explains the uncontrolled proliferation, which ultimately progresses to cervical cancer. However, little is known about the specific factors that promote or inhibit the malignant transformation. Since a portion of HPV infections never evolves into cancer, the identification of discriminatory factors would be an important step towards the development of prognostic and therapeutic tools. Based on this notion, we investigated whether an isoform of BARD1, BARD1δ, which we found frequently expressed in cancer cells, plays a role in the malignant transformation of cervical epithelial cells. Using immunohystochemistry staining on tissue sections and RT-PCR we found that BARD1δ is expressed in proliferating cervical cancer cells, while normal BARD1 is repressed. BARD1 might be a driver of carcinogenesis and might be a critical event in the progression from hyper-proliferating cervical epithelium after HPV infection to cervical cancer. Together, our results show that an isoform of BARD1, BARD1δ, is increasingly expressed in precancerous stages of cervical cancer and the specific inhibition of BARD1δ may represent a novel approach for therapeutic interventions to prevent cervical carcinogenesis after HPV infection. Citation Format: Maxim Pilyugin, Pierre-Alain André, Nicole Concin, Gerda Hofstetter, Irmgard Irminger-Finger. Expression of BARD1δ induces genetic instability and is associated with cervical carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1417. doi:10.1158/1538-7445.AM2017-1417

Abstract 1170: Notch pathway is overexpressed and is a therapeutic target in clear cell renal cancer

Abstract Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. We conducted a transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls and observed an overexpression of NOTCH ligands [JAGGED1, JAGGED2) and Delta like (DLL3) family of ligands] and receptors (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) in tumor tissues. Examination of the TCGA RNA-Seq dataset also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival. Parallel high resolution DNA methylation and copy number analysis with the HELP assay demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligands, JAGGED1 was overexpressed and associated with loss of methylation of its intronic enhancer. The other NOTCH ligand, JAGGED2 was overexpressed in and associated with gene amplification in distinct CCRCC samples. To test the causality, we transgenically expressed the intracellular domain of NOTCH1 in mice renal tubules with tubule specific deletion of VHL. The Kspcre VHLf/f ICNotch1 mice exhibited dysplastic hyperproliferation of tubular epithelial cells confirming the procarcinogenic role of NOTCH in vivo. Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain. Finally, treatment with clinical, gamma secretase inhibitor, LY3039478, led to inhibition of CCRCC cells in vitro and in vivo in CCRCC xenografts. In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target. Citation Format: Tushar D. Bhagat, Yiyu Zou, Shizeng Huang, Jihwan Park, Matthew B. Palmer, Caroline Hu, Weijuan Li, Niraj Shenoy, Orsolya Giricz, Yiting Yu, Yi-An Ko, María Concepción Izquierdo, Esther Park, Nishanth Vallumsetla, Remi Laurence, Robert Lopez, Masako Suzuki, James Pullman, Justin Kaner, Benjamin Gartrell, A. Ari Hakimi, John Greally, Bharvin Patel, Karim Benhadji, Kith Pradhan, Amit Verma, Katalin Susztak. Notch pathway is overexpressed and is a therapeutic target in clear cell renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1170. doi:10.1158/1538-7445.AM2017-1170

The changes in glucose metabolism and cell proliferation in the kidneys of polycystic kidney disease mini-pig models

The pathogenic mechanism of polycystic kidney disease (PKD) is unclear. Abnormal glucose metabolism is maybe involved in hyper-proliferation of renal cyst epithelial cells. Mini-pigs are more similar to humans than rodents and therefore, are an ideal large animal model. In this study, for the first time, we systematically investigated the changes in glucose metabolism and cell proliferation signaling pathways in the kidney tissues of chronic progressive PKD mini-pig models created by knock-outing PKD1gene.The results showed that in the kidneys of PKD mini-pigs, the glycolysis is increased and the expressions of key oxidative phosphorylation enzymes Complexes I and IV significantly decreased. The activities of mitochondrial respiration chain Complexes I and IV significantly decreased; the phosphorylation level of key metabolism-modulating molecule AMP-activated protein kinase (AMPK) significantly decreased; and the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) signaling pathway are activated obviously.This study showed that in the kidneys of PKD mini-pigs, the level of glycolysis significantly increased, oxidative phosphorylation significantly decreased, and cell proliferation signaling pathways significantly activated, suggesting that metabolic changes in PKD may result in the occurrence and development of PKD through the activation of proliferation signaling pathways.

Notch Pathway Is Activated via Genetic and Epigenetic Alterations and Is a Therapeutic Target in Clear Cell Renal Cancer

Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. Transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls revealed overexpression of NOTCH ligands and receptors in tumor tissues. Examination of the TCGA RNA-seq data set also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival. Parallel DNA methylation and copy number analysis demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligand JAGGED1 was overexpressed and associated with loss of CpG methylation of H3K4me1-associated enhancer regions. JAGGED2 was also overexpressed and associated with gene amplification in distinct CCRCC samples. Transgenic expression of intracellular NOTCH1 in mice with tubule-specific deletion of VHL led to dysplastic hyperproliferation of tubular epithelial cells, confirming the procarcinogenic role of NOTCH in vivo Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression, and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain. Treatment with the γ-secretase inhibitor LY3039478 led to inhibition of CCRCC cells in vitro and in vivo In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target.

Intermittent fasting prompted recovery from dextran sulfate sodium-induced colitis in mice.

Fasting-refeeding in mice induces transient hyperproliferation of colonic epithelial cells, which is dependent on the lactate produced as a metabolite of commensal bacteria. We attempted to manipulate colonic epithelial cell turnover with intermittent fasting to prompt recovery from acute colitis. Acute colitis was induced in C57BL/6 mice by administration of dextran sulfate sodium in the drinking water for 5 days. From day 6, mice were fasted for 36 h and refed normal bait, glucose powder, or lactylated high-amylose starch. On day 9, colon tissues were subjected to analysis of histology and cytokine expression. The effect of lactate on the proliferation of colonocytes was assessed by enema in vivo and primary culture in vitro. Intermittent fasting resulted in restored colonic crypts and less expression of interleukin-1β and interleukin-17 in the colon than in mice fed ad libitum. Administration of lactate in the colon at refeeding time by enema or by feeding lactylated high-amylose starch increased the number of regenerating crypts. Addition of lactate but not butyrate or acetate supported colony formation of colonocytes in vitro. In conclusion, intermittent fasting in the resolution phase of acute colitis resulted in better recovery of epithelial cells and reduced inflammation.

Murine Double Minute-2 Inhibition Ameliorates Established Crescentic Glomerulonephritis

Rapidly progressive glomerulonephritis is characterized by glomerular necroinflammation and crescent formation. Its treatment includes unspecific and toxic agents; therefore, the identification of novel therapeutic targets is required. The E3-ubiquitin ligase murine double minute (MDM)-2 is a nonredundant element of NF-κB signaling and the negative regulator of tumor suppressor gene TP53-mediated cell cyclearrest and cell death. We hypothesized that the MDM2 would drive crescentic glomerulonephritis byNF-κB-dependent glomerular inflammation and by p53-dependent parietal epithelial cell hyperproliferation. Indeed, the pre-emptive MDM2 blockade by nutlin-3a ameliorated all aspects of crescentic glomerulonephritis. MDM2 inhibition had identical protective effects in Trp53-deficient mice, with the exception of crescent formation, which was not influenced by nutlin-3a treatment. Invitro experiments confirmed the contribution of MDM2 for induction of NF-κB-dependent cytokines in murine glomerular endothelial cells and for p53-dependent parietal epithelial cell proliferation. To evaluate MDM2 blockade as a potential therapeutic intervention in rapidly progressive glomerulonephritis, we treated mice with established glomerulonephritis with nutlin-3a. Delayed onset of nutlin-3a treatment was equally protective as the pre-emptive treatment in abrogating crescentic glomerulonephritis. Together, the pathogenic effects of MDM2 are twofold, that is, p53-independent NF-κB activation increasing intraglomerular inflammation and p53-dependent parietal epithelial cell hyperplasia and crescent formation. We therefore propose MDM2 blockade as a potential novel therapeutic strategy in rapidly progressive glomerulonephritis.

The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability.

SummaryRegulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs) during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs) depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability.

Hyperproliferation in nasal polyposis tissues is not associated with somatic genomic instability

Nasal polyps (NPs) are abnormal lesions that arise from any portion of the nasal mucosa or paranasal sinuses within the nasal cavities. They are characterized by the formation of inflammatory swellings of unstable respiratory mucosa, which protrude into the nasal cavity. The causes and the pathogenesis of NP development are not well understood. The current and principal hypothesis is that NPs result from allergy and inflammation, which cause an unstable respiratory mucosa with increased epithelial cell proliferation and epithelial morphologic changes. To understand if epithelial cell hyperproliferation can lead to acquired genomic instability and if nasal polyposis is a consequence of acquired chromosomal alterations in hyperproliferative nasal epithelia. To test this hypothesis and to perform a survey of chromosomal aberrations in nasal polyps, we applied array comparative genome hybridization (aCGH; 1 Mb resolution, made of 2464 bacterial artificial chromosome (BAC), P1 derived artificial chromosome (PAC), and P1 clones spanning the whole human genome) to deoxyribonucleic acid (DNA) obtained from the NPs of nine patients. The patients recruited in this study had been analyzed for Ki-67 expression using the MIB-1 antibody as a marker of proliferation and were chosen to represent a wide range of hyperproliferative status. After data analysis, no chromosomal aberrations were detected by aCGH. Our data do not support the hypothesis that nasal polyposis is a consequence of acquired chromosomal alterations in hyperproliferative nasal epithelia.

Activation of NF-κB and AP-1 Mediates Hyperproliferation by Inducing β-Catenin and c-Myc in Helicobacter pylori-Infected Gastric Epithelial Cells.

PurposeIn the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as β-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of β-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells.Materials and MethodsGastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori.ResultsH. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (β-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells.ConclusionH. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells.

Evidence for a "Pathogenic Triumvirate" in Congenital Hepatic Fibrosis in Autosomal Recessive Polycystic Kidney Disease.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe monogenic disorder that occurs due to mutations in the PKHD1 gene. Congenital hepatic fibrosis (CHF) associated with ARPKD is characterized by the presence of hepatic cysts derived from dilated bile ducts and a robust, pericystic fibrosis. Cyst growth, due to cyst wall epithelial cell hyperproliferation and fluid secretion, is thought to be the driving force behind disease progression. Liver fibrosis is a wound healing response in which collagen accumulates in the liver due to an imbalance between extracellular matrix synthesis and degradation. Whereas both hyperproliferation and pericystic fibrosis are hallmarks of CHF/ARPKD, whether or not these two processes influence one another remains unclear. Additionally, recent studies demonstrate that inflammation is a common feature of CHF/ARPKD. Therefore, we propose a “pathogenic triumvirate” consisting of hyperproliferation of cyst wall growth, pericystic fibrosis, and inflammation which drives CHF/ARPKD progression. This review will summarize what is known regarding the mechanisms of cyst growth, fibrosis, and inflammation in CHF/ARPKD. Further, we will discuss the potential advantage of identifying a core pathogenic feature in CHF/ARPKD to aid in the development of novel therapeutic approaches. If a core pathogenic feature does not exist, then developing multimodality therapeutic approaches to target each member of the “pathogenic triumvirate” individually may be a better strategy to manage this debilitating disease.

PP8 - Crosstalk of JAK1/STAT3 and WNT/β-catenin in Helicobacter pylori-induced hyper-proliferation in gastric epithelial cells: Effect of antioxidant lycopene

Helicobacter pylori (H. pylori) infection, as a risk factor for gastric cancer, is associated with hyper-proliferation and oncogene expression in gastric epithelial cells. Previously, we showed that reactive oxygen species (ROS) mediate activation of JAK/STAT signaling and hyper-proliferation in H. pylori–infected gastric epithelial cells. Since JAK signaling is known to mediate Wnt/β-catenin pathway in various cells, we investigated the relation of ROS, JAK/STAT activation, and Wnt/β-catenin pathway for hyper-proliferation in H. pylori-infected gastric epithelial AGS cells using an antioxidant lycopene. Lycopene, a bright red carotenoid pigment in fruits and vegetables, has anticancer activity in many types of cancers. As a result, H. pylori induced hyper-proliferation in parallel with induction and nuclear translocation of β-catenin. For Wnt/β-catenin signaling, H. pylori decreased the level of adenomatous polyposis coli (APC), a component of the destruction complex targeting β-catenin, and induced β-catenin target genes (cyclin-E, c-myc) in AGS cells. H. pylori activated GSK3β, but reduced APC and Axin in AGS cells. For JAK/STAT signaling, Jak1 and Stat3 were activated in H. pylori-infected AGS cells. Jak1 inhibitor AG490 decreased levels of β-catenin and its target genes (cyclin-E, c-myc) and increased the level of APC in H. pylori-infected cells. Lycopene inhibited H. pylori-induced ROS production, activation of JAK1/STAT3, reduction of APC/AXIN, and induction of β-catenin and its target genes (cyclin-E, c-myc), and hyper-proliferation in AGS cells. In conclusion, lycopene inhibits H. pylori-induced hyper-proliferation by suppressing ROS-mediated activation of JAK/STAT and WNT/β-catenin signaling in gastric epithelial cells.

Helicobacter pylori Alters the Expression of Circadian Clock Components PER2 and BMAL1 During Infection

The circadian clock plays a fundamental role in gastrointestinal (GI) physiology. Moreover, disruption of key components of the molecular oscillator that generates circadian rhythms, such as Per2 and Bmal1, promotes GI tumorigenesis. Glycogen synthase kinase-3β (GSK-3β), a known regulator of epithelial cell proliferation, also regulates PER2. Here we test the hypothesis that Helicobacter pylori (H. pylori)-induced epithelial cell proliferation is dependent on the inactivation of GSK-3β leading to the activation of BMAL1. In vivo, H. pylori induced a significant upregulation of BMAL1 expression within the gastric fundus of infected mice C57BL/6 that correlated with a significant increase in epithelial proliferation and the development of metaplasia. Bioluminescent gastric-derived organoids generated from PER2::LUCIFERASE (PER2::LUC) mice showed robust circadian rhythms. H. pylori infection of PER2::LUC mouse-derived gastric organoids resulted in decreased PER2::LUC activity. Robust circadian rhythmicity of clock genes BMAL1 and PER2 were observed in human fundic-derived gastric organoids (hFGOs). Phosphorylation (inactivation) of GSK-3β showed circadian changes in hFGOs over 52 hours. H. pylori infection of hFGOs resulted in sustained GSK-3β phosphorylation, increased BMAL1 protein expression and epithelial cell hyperproliferation when compared to uninfected controls. These results indicate that H. pylori alters the expression of circadian clock components during infection in association with epithelial cell hyperproliferation and metaplasia.

Intestinal microbial dysbiosis and colonic epithelial cell hyperproliferation by dietary α-mangostin is independent of mouse strain.

Beverages and supplements prepared from mangosteen fruit are claimed to support gut health and immunity, despite the absence of supporting evidence from clinical trials. We recently reported that α-mangostin (α-MG), the most abundant xanthone in mangosteen fruit, altered the intestinal microbiome, promoted dysbiosis, and exacerbated colitis in C57BL/6J mice. The objective of this study was to determine whether induction of dysbiosis by dietary α-MG is limited to the C57BL/6J strain or represents a more generic response to chronic intake of the xanthone on the gut microbiota of mice. C3H, Balb/c, Nude FoxN1nu, and C57BL/6J mice, each demonstrating unique microbiomes, were fed standard diet or diet containing 0.1% α-MG for four weeks. Dietary α-MG significantly altered the cecal and colonic microbiota in all four strains of mice, promoting a reduction in generally assumed beneficial bacterial groups while increasing the abundance of pathogenic bacteria. Consumption of α-MG was associated with reduced abundance of Firmicutes and increased abundance of Proteobacteria. The abundance of Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae was reduced in α-MG-fed mice, while that of Enterobacteriaceae and Enterococcaceae was increased. Dietary α-MG also was associated with increased proliferation of colonic epithelial cells, infiltration of immune cells, infiltration of immune cells and increased fluid content in stool. These results suggest that ingestion of pharmacologic doses of xanthones in mangosteen-containing supplements may adversely alter the gut microbiota and should be used with caution.

Transforming growth factor β1 increases p27 levels via synthesis and degradation mechanisms in the hyperproliferative gastric epithelium in rats.

Throughout postnatal development, the gastric epithelium expresses Transforming Growth Factor beta1 (TGFβ1), but it is also exposed to luminal peptides that are part of milk. During suckling period, fasting promotes the withdrawal of milk-born molecules while it stimulates gastric epithelial cell proliferation. Such response can be reversed by exogenous TGFβ1, as it directly affects cell cycle through the regulation of p27 levels. We used fasting condition to induce the hyperproliferation of gastric epithelial cells in 14-day-old Wistar rats, and evaluated the effects of TGFβ1 gavage on p27 expression, phosphorylation at threonine 187 (phospho-p27Thr187) and degradation. p27 protein level was reduced during fasting when compared to suckling counterparts, while phospho-p27Thr187/p27 ratio was increased. TGFβ1 gavage reversed this response, which was confirmed through immunostaining. By using a neutralizing antibody against TGFβ1, we found that it restored the p27 and phosphorylation levels detected during fasting, indicating the specific role of the growth factor. We noted that neither fasting nor TGFβ1 changed p27 expression, but after cycloheximide administration, we observed that protein synthesis was influenced by TGFβ1. Next, we evaluated the capacity of the gastric mucosa to degrade p27 and we recorded a higher concentration of the remaining protein in pups treated with TGFβ1, suggesting augmented stability under this condition. Thus, we showed for the first time that luminal TGFβ1 increased p27 levels in the rat gastric mucosa by up- regulating translation and reducing protein degradation. We concluded that such mechanisms might be used by rapidly proliferating cells to respond to milk-born TGFβ1 and food restriction.