Reg4 Interacts with CD44 to Regulate Proliferation and Stemness of Colorectal and Pancreatic Cancer Cells.

Regenerating Gene 4 (Reg4) is highly upregulated in gastrointestinal (GI) malignancies including colorectal and pancreatic cancers. Numerous studies demonstrated an association between higher Reg4 expression and tumor aggressiveness, intrinsic resistance to apoptotic death, and poor outcomes from GI malignancies. However, the precise receptor and underlying signaling mechanism have remained unknown. Although we previously reported a Reg4-mediated induction of EGFR activity in colorectal cancer cells, a direct interaction between Reg4 and EGFR was not observed. This study is focused on identifying the cell surface binding partner of Reg4 and dissecting its role in colorectal cancer and pancreatic cancer growth and stem cell survival. In vitro models of human colorectal cancer and pancreatic cancer were used to evaluate the results. Results of this study find: (i) Reg4 interacts with CD44, a transmembrane protein expressed by a population of colorectal cancer and pancreatic cancer cells; (ii) Reg4 activates regulated intramembrane proteolysis of CD44 resulting in γ-secretase-mediated cleavage and release of the CD44 intracytoplasmic domain (CD44ICD) that functions as a transcriptional activator of D-type cyclins involved in the regulation of cancer cell proliferation and Klf4 and Sox2 expression involved in regulating pluripotency of cancer stem cells; and (iii) Reg4 significantly increases colorectal cancer and pancreatic cancer cell proliferation and their clonogenic potential in stem cell assays. IMPLICATIONS: These results suggest that pro-proliferative and pro-stemness effects of Reg4 are mediated through γ-secretase-mediated CD44/CD44ICD signaling, hence strategies to disrupt Reg4-CD44-γ-secretase-CD44ICD signaling axis may increase cancer cell susceptibility to chemo- and radiotherapeutics.

Reg4 and its downstream transcriptional activator CD44ICD in stage II and III colorectal cancer.

Reg4 is highly expressed in gastrointestinal malignancies and acts as a mitogenic and pro-invasive factor. Our recent works suggest that Reg4 binds with CD44 and induces its proteolytic cleavage to release intra-cytoplasmic domain of CD44 (CD44ICD). The goal of this study is to demonstrate clinical significance of the Reg4-CD44/CD44ICD pathway in stage II/III colon cancer and its association with clinical parameters of aggression. We constructed a tissue microarray (TMA) of 93 stage II/III matched colon adenocarcinoma patients, 23 with recurrent disease. The TMA was immunohistochemically stained for Reg4, CD44, and CD44ICD proteins and analyzed to identify associations with tumor characteristics, recurrence and overall survival. The TMA data analysis showed a significant correlation between Reg4 and CD44 (r2 = 0.23, P = 0.028), CD44 and CD44ICD (r2 = 0.36, p = 0.0004), and Reg4 and CD44ICD (r2 = 0.45, p ≤ 0.0001). Reg4 expression was associated with larger tumor size (r2 = 0.23, p = 0.026). Although, no association was observed between Reg4, CD44, or CD44ICD expression and disease recurrence, Reg4-positive patients had a median survival of 4 years vs. 7 years for Reg4-negative patients (p = 0.04) in patients who recurred. Inhibition of the Reg4-CD44/CD44ICD pathway may be a future therapeutic target for colon cancer patients.

Quality metrics of screening colonoscopies performed by PAs.

BACKGROUND: Many communities face a shortage of qualified endoscopists. Training physician assistants (PAs) to perform colonoscopies can expand the availability of colorectal cancer screening. This study examined screening colonoscopy metrics and quality indicators among gastroenterologists, supervised PAs, and gastroenterology fellows. METHODS: Consecutive patients undergoing average-risk screening colonoscopy were stratified into one of three groups by endoscopist type. Procedure and pathology reports were reviewed for the technical performance and quality metrics of the providers. RESULTS: PAs performed comparably to gastroenterologists in technical performance and quality metrics, and demonstrated higher cecal intubation rates than their gastroenterologist colleagues. Comparisons of attending physicians and PAs grouped by years of experience also did not show notable differences in performance. CONCLUSIONS: In a supervised practice, PAs performed on par with their gastroenterology colleagues on established colonoscopy quality indicators. Following proper training, PAs can be employed in the provision of screening colonoscopy.

IDO1 and Kynurenine Pathway Metabolites Activate PI3K-Akt Signaling in the Neoplastic Colon Epithelium to Promote Cancer Cell Proliferation and Inhibit Apoptosis.

The tryptophan-metabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) is frequently overexpressed in epithelial-derived malignancies, where it plays a recognized role in promoting tumor immune tolerance. We previously demonstrated that the IDO1-kynurenine pathway (KP) also directly supports colorectal cancer growth by promoting activation of ß-catenin and driving neoplastic growth in mice lacking intact adaptive immunity. In this study, we sought to delineate the specific role of epithelial IDO1 in colon tumorigenesis and define how IDO1 and KP metabolites interact with pivotal neoplastic signaling pathways of the colon epithelium. We generated a novel intestinal epithelial-specific IDO1 knockout mouse and utilized established colorectal cancer cell lines containing ß-catenin-stabilizing mutations, human colorectal cancer samples, and human-derived epithelial organoids (colonoids and tumoroids). Mice with intestinal epithelial-specific knockout of IDO1 developed fewer and smaller tumors than wild-type littermates in a model of inflammation-driven colon tumorigenesis. Moreover, their tumors exhibited reduced nuclear ß-catenin and neoplastic proliferation but increased apoptosis. Mechanistically, KP metabolites (except kynurenic acid) rapidly activated PI3K-Akt signaling in the neoplastic epithelium to promote nuclear translocation of ß-catenin, cellular proliferation, and resistance to apoptosis. Together, these data define a novel cell-autonomous function and mechanism by which IDO1 activity promotes colorectal cancer progression. These findings may have implications for the rational design of new clinical trials that exploit a synergy of IDO1 inhibitors with conventional cancer therapies for which Akt activation provides resistance such as radiation.Significance: This study identifies a new mechanistic link between IDO1 activity and PI3K/AKT signaling, both of which are important pathways involved in cancer growth and resistance to cancer therapy.

Reg4-induced mitogenesis involves Akt-GSK3ß-ß-Catenin-TCF-4 signaling in human colorectal cancer.

Upregulation of regenerating gene 4 (Reg4) is observed in many human gastrointestinal malignancies including colorectal cancer (CRC). We previously reported a Reg4-mediated induction of epidermal growth factor receptor-Akt-AP1 signaling regulating CRC cell apoptosis. However, the role of Reg4 in the regulation of CRC cell division is poorly understood. This study tests the hypothesis that Reg4 induces Akt-GSK3ß-ß-Catenin-TCF-4 signaling to regulate CRC cell division. In vitro models of human CRC were used to determine the role of Reg4 in regulation of CRC cell division. Cell cycle studies demonstrated that Reg4 treatment significantly decreased CRC cell number in G1 phase and increased in G2 phase. Subsequently Reg4 significantly increased the mitotic index of CRC cells. As assessed by real-time RT-PCR and Western blot analyses, Reg4 significantly increased the expression of cell cycle regulatory genes Cyclin D1 and D3, and associated Cyclin-dependent kinases (CDK4 and CDK6). Reg4-mediated increase in these genes involved a pathway that included an induced Akt activity by increasing phosphorylation of Thr308 and Ser473, a reduced glycogen synthase kinase 3ß (GSK-3ß) activity by increasing phosphorylation of Ser9, an induced nuclear translocation of ß-Catenin by decreasing phosphorylation of Ser33/37/Thr41, and an increased TCF-4 transcriptional activity. Furthermore, antagonism of Reg4-signaling using Reg4-specific mAbs (2H6 and 3E5) and Akt inhibitor significantly decreased, whereas agonism using GSK-3ß antagonist (SB216763) significantly increased mitotic index and proliferation of CRC cells. These results identify Reg4 as a key regulator of the CRC cell division and proliferation, hence a potential target of human CRC treatment.

RNA binding protein CUGBP2/CELF2 mediates curcumin-induced mitotic catastrophe of pancreatic cancer cells.

BACKGROUND: Curcumin inhibits the growth of pancreatic cancer tumor xenografts in nude mice; however, the mechanism of action is not well understood. It is becoming increasingly clear that RNA binding proteins regulate posttranscriptional gene expression and play a critical role in RNA stability and translation. Here, we have determined that curcumin modulates the expression of RNA binding protein CUGBP2 to inhibit pancreatic cancer growth. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that curcumin treated tumor xenografts have a significant reduction in tumor volume and angiogenesis. Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells. This was further confirmed by increased phosphorylation of checkpoint kinase 2 (Chk2) protein coupled with higher levels of nuclear cyclin B1 and Cdc-2. Curcumin increased the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA, but protein levels were lower. Furthermore, curcumin increased the expression of RNA binding proteins CUGBP2/CELF2 and TIA-1. CUGBP2 binding to COX-2 and VEGF mRNA was also enhanced, thereby increasing mRNA stability, the half-life changing from 30 min to 8 h. On the other hand, silencer-mediated knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF even with curcumin treatment. COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher. CONCLUSION/SIGNIFICANCE: Curcumin inhibits pancreatic tumor growth through mitotic catastrophe by increasing the expression of RNA binding protein CUGBP2, thereby inhibiting the translation of COX-2 and VEGF mRNA. These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers.

NOD2 status and human ileal gene expression.

BACKGROUND: NOD2 single nucleotide polymorphisms have been associated with increased risk of ileal Crohn's disease (CD). This exploratory study was conducted to compare ileal mucosal gene expression in CD patients with and without NOD2 risk alleles. METHODS: Ileal samples were prospectively collected from 18 nonsmoking CD patients not treated with anti-TNF-α biologics and 9 nonsmoking control patients without inflammatory bowel disease undergoing initial resection and genotyped for the 3 major NOD2 risk alleles (Arg702Trp, Gly908Arg, Leu1007fs). Microarray analysis was performed in samples from 4 NOD2(R) (at least 1 risk allele) CD patients, 4 NOD2(NR) (no risk alleles) CD patients, and 4 NOD2(NR) controls. Candidate genes selected by significance analysis of microarrays (SAM) were confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays of all the samples. RESULTS: SAM detected upregulation of 18 genes in affected ileum in NOD2(R) compared to NOD2(NR) CD patients, including genes related to lymphocyte activation. SAM also detected altered ileal gene expression in unaffected NOD2(NR) ileal mucosal CD samples compared to NOD2(NR) control samples. qRT-PCR conducted on all the samples confirmed that increased CD3D expression in affected samples was associated with NOD2(R) status, and that increased MUC1, DUOX2, DMBT1 and decreased C4orf7 expression in unaffected samples was associated with CD, independent of NOD2 status. CONCLUSIONS: The results support the concept that NOD2 risk alleles contribute to impaired regulation of inflammation in the ileum. Furthermore, altered ileal gene expression, independent of NOD2 status, is detected in the unaffected proximal margin of resected ileum from CD patients.

Reg IV regulates normal intestinal and colorectal cancer cell susceptibility to radiation-induced apoptosis.

BACKGROUND & AIMS: Regenerating (Reg) gene IV is predominantly expressed in gastrointestinal cells and highly up-regulated in many gastrointestinal malignancies, including colorectal cancer (CRC). Human CRC cells expressing higher levels of Reg IV gene and its protein product (Reg IV) are resistant to conventional therapies, including irradiation (IR). However, the underlying mechanism is not well defined. METHODS: A murine model of IR-induced intestinal injury and in vitro and in vivo models of human CRC were used to determine the role of Reg IV in regulation of normal intestinal and colorectal cancer cell susceptibility to IR-induced apoptosis. RESULTS: Treatments of recombinant human Reg IV (rhR4) protein protected normal intestinal crypt cells from IR-induced apoptosis by increasing the expression of antiapoptotic genes Bcl-2, Bcl-XL, and survivin. However, overexpression of Reg IV in human CRC cells was associated with increased resistance to IR-induced apoptosis. Therefore, we used antagonism of Reg IV as a tool to increase CRC cell susceptibility to IR-induced cell death. Two complementary approaches using specific monoclonal antibodies and small interfering RNAs were tested in both in vitro and in vivo models of human CRC. Both approaches resulted in increased apoptosis and decreased cell proliferation, leading to decreased tumor growth and increased animal survival. Furthermore, these approaches increased CRC cell susceptibility to IR-induced apoptosis. CONCLUSIONS: These results implicate Reg IV as an important modulator of gastrointestinal cell susceptibility to IR; hence, it is a potential target for adjunctive treatments for human CRC and other gastrointestinal malignancies.

Loss of p21Waf1/Cip1/Sdi1 enhances intestinal stem cell survival following radiation injury.

The microcolony assay following gamma irradiation (IR) is a functional assay of intestinal stem cell fate. The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) (p21) regulates cell cycle arrest following DNA damage. To explore the role of p21 on stem cell fate, we examined the effects of p21 deletion on intestinal crypt survival following IR and expression of the stem/progenitor cell marker Musashi-1 (Msi-1) and the antiapoptotic gene survivin. Intestinal stem cell survival in adult wild-type (WT) and p21(-/-) mice was measured using the microcolony assay. Msi-1, p21, and survivin mRNA were measured using real-time PCR and immunohistochemical analysis. Laser capture microdissection (LCM) was used to isolate mRNA from the crypt stem cell zone. No differences in radiation-induced apoptosis were observed between WT and p21(-/-) mice. However, increased crypt survival (3.0-fold) was observed in p21(-/-) compared with WT mice 3.5 days after 13 Gy. Msi-1 and survivin mRNA were elevated 12- and 7.5-fold, respectively, in LCM-dissected crypts of p21(-/-) compared with WT mice. In conclusion, deletion of p21 results in protection of crypt stem/progenitor cells from IR-induced cell death. Furthermore, the increase in crypt survival is associated with increased numbers of Msi-1- and survivin-expressing cells in regenerative crypts.

REG1A expression is a prognostic marker in colorectal cancer and associated with peritoneal carcinomatosis.

By expression profiling of early staged colon carcinomas, we found regenerating islet-derived 1 alpha (REG1A) to be upregulated in patients with an unfavorable clinical outcome. For validation, REG1A expression was quantified in another colorectal cancer (CRC) patient cohort by Taqman PCR. Aside from tumor and normal tissue from 63 nonpretreated CRC patients, 31 mucosa biopsies from healthy individuals as well as 22 adenomas were included in the investigation. REG1A was significantly upregulated in tumor specimens (p < 0.001) and adenoma (p < 0.01) as compared to normal colorectal tissue. REG1A expression in normal peritumoral tissue in turn proved to be significantly elevated compared to mucosa from healthy individuals (p < 0.01). Determination of REG1A expression might be useful for early tumor diagnosis with a sensitivity of 90.6%, and a specificity of 77.9%. REG1A expression was significantly increased in tumors with peritoneal carcinomatosis (p < 0.01). Moreover, REG1A turned out to be a significant predictor of disease-free survival (p < 0.05). In conclusion, we present evidence that REG1A is a molecular marker of prognostic value and is associated with peritoneal carcinomatosis in CRC. REG1A turned out to be already significantly raised in peritumoral normal tissue compared to mucosa from healthy individuals, suggesting a molecular field effect of secreted REG1A.

Knockdown of RNA binding protein musashi-1 leads to tumor regression in vivo.

BACKGROUND & AIMS: In the gut, tumorigenesis is thought to arise from the stem cell population located near the base of intestinal and colonic crypts. The RNA binding protein musashi-1 (Msi-1) is a putative intestinal and progenitor/stem cell marker. Msi-1 expression is increased during rat brain development and in APC(min/+) mice tumors. This study examined a potential role of Msi-1 in tumorigenesis. METHODS: Msi-1 small interfering RNA (siRNA) was administered as a liposomal preparation to HCT116 colon adenocarcinoma xenografts in athymic nude mice and tumor volume was measured. Cell proliferation was assessed by hexosaminidase and 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide MTT assays. siRNA-transfected cells were subjected to 12 Gy gamma-irradiation. Apoptosis was assessed by immunoreactive activated caspase-3 and mitosis was assessed by phosphorylated histone H3 staining. The tumor xenografts were stained similarly for phosphorylated histone H3, activated caspase-3, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, Notch-1, and p21(WAF1). Furthermore, siRNA-transfected cells were subjected to cell-cycle analysis and Western blot analyses for Notch-1 and p21(WAF1). RESULTS: Knockdown of Msi-1 resulted in tumor growth arrest in xenografts, reduced cancer cell proliferation, and increased apoptosis alone and in combination with radiation injury. siRNA-mediated reduction of Msi-1 lead to mitotic catastrophe in tumor cells. Moreover, there was inhibition of Notch-1 and up-regulation of p21(WAF1) after knockdown of Msi-1. CONCLUSIONS: Our results show the involvement of Msi-1 in cancer cell proliferation, inhibition of apoptosis, and mitotic catastrophe, suggesting an important potential mechanism for its role in tumorigenesis.

Gastrin-mediated interleukin-8 and cyclooxygenase-2 gene expression: differential transcriptional and posttranscriptional mechanisms.

BACKGROUND & AIMS: Gastrin induces the expression of cyclooxygenase (COX)-2 and interleukin (IL)-8; however, the mechanism(s), especially in gastric epithelial cells, is not well understood. Here, we have determined the intracellular mechanisms mediating gastrin-dependent gene expression. METHODS: AGS-E human gastric cancer cell line stably expressing cholecystokinin-2 receptor was treated with amidated gastrin-17. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were performed to determine COX-2 and IL-8 expression and Akt, Erk, and p38 phosphorylation. Gene promoter activity was determined by luciferase assay. Electrophoretic mobility shift assay analysis was performed for nuclear factor kappaB (NF-kappaB) and activator protein-1 activity. RNA stability was determined after actinomycin D treatment. HuR localization was determined by immunocytochemistry. RESULTS: Gastrin induced COX-2 and IL-8 expression in AGS-E cells, which was inhibited by phosphatidylinositol 3' kinase (PI3K) and p38 inhibitors. Gastrin-mediated Akt activation was observed to be downstream of p38. IL-8 expression was dependent on COX-2-mediated prostaglandin E(2) synthesis. In the presence of an NF-kappaB inhibitor MG132, IL-8 transcription was inhibited, but not that of COX-2. This was confirmed after knockdown of the p65 RelA subunit of NF-kappaB. Further studies showed that COX-2 gene transcription is regulated by activator protein-1. Gastrin increased the stability of both COX-2 and IL-8 messenger RNA (mRNA) in a p38-dependent manner, the half-life increasing from 31 minutes to 8 hours and approximately 4 hours, respectively. Gastrin, through p38 activity, also enhanced HuR expression, nucleocytoplasmic translocation, and enhanced COX-2 mRNA binding. CONCLUSIONS: Gastrin differentially induces COX-2 and IL-8 expression at the transcriptional and posttranscriptional levels by PI3K and p38 mitogen-activated protein kinase pathways, respectively.

Diphenyl difluoroketone: a curcumin derivative with potent in vivo anticancer activity.

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was reported to inhibit proliferation of a variety of cancer cells in vitro. However, the efficacy and in vivo mechanism of action of EF24 in gastrointestinal cancer cells have not been investigated. Here, we assessed the in vivo therapeutic effects of EF24 on colon cancer cells. Using hexosaminidase assay, we determined that EF24 inhibits proliferation of HCT-116 and HT-29 colon and AGS gastric adenocarcinoma cells but not of mouse embryo fibroblasts. Furthermore, the cancer cells showed increased levels of activated caspase-3 and increased Bax to Bcl-2 and Bax to Bcl-xL ratios, suggesting that the cells were undergoing apoptosis. At the same time, cell cycle analysis showed that there was an increased number of cells in the G(2)-M phase. To determine the effects of EF24 in vivo, HCT-116 colon cancer xenografts were established in nude mice and EF24 was given i.p. EF24 significantly suppressed the growth of colon cancer tumor xenografts. Immunostaining for CD31 showed that there was a lower number of microvessels in the EF24-treated animals coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA and protein expression. Western blot analyses also showed decreased AKT and extracellular signal-regulated kinase activation in the tumors. Taken together, these data suggest that the novel curcumin-related compound EF24 is a potent antitumor agent that induces caspase-mediated apoptosis during mitosis and has significant therapeutic potential for gastrointestinal cancers.

Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis.

BACKGROUND: Sargramostim, granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, stimulates cells of the intestinal innate immune system. Clinical trials show that sargramostim induces clinical response and remission in patients with active Crohn's disease. To study the mechanism, we examined the effects of GM-CSF in the dextran sulfate sodium (DSS)-induced acute colitis model. We hypothesized that GM-CSF may work through effects on dendritic cells (DCs). METHODS: Acute colitis was induced in Balb/c mice by administration of DSS in drinking water. Mice were treated with daily GM-CSF or phosphate-buffered saline (PBS). To probe the role of plasmacytoid DCs (pDCs) in the response to GM-CSF, we further examined the effects of monoclonal antibody 440c, which is specific for a sialic acid-binding immunoglobulin (Ig)-like lectin expressed on pDCs. RESULTS: GM-CSF ameliorates acute DSS-induced colitis, resulting in significantly improved clinical parameters and histology. Microarray analysis showed reduced expression of proinflammatory genes including TNF-alpha and IL1-beta; the results were further confirmed by real-time reverse-transcriptase polymerase chain reaction and serum Bio-plex analysis. GM-CSF treatment significantly expands pDCs and type 1 IFN production. Administration of mAb 440c completely blocked the therapeutic effect of GM-CSF. GM-CSF is also effective in RAG1(-/-) mice, demonstrating activity-independent effects on T and B cells. IFN-beta administration mimics the therapeutic effect of GM-CSF in DSS-treated mice. GM-CSF increases systemic and mucosal type 1 IFN expression and exhibits synergy with pDC activators, such as microbial cytosine-phosphate-guanosine (CpG) DNA. CONCLUSIONS: GM-CSF is effective in the treatment of DSS colitis in a mechanism involving the 440c(+) pDC population.

Expression of the genes dual oxidase 2, lipocalin 2 and regenerating islet-derived 1 alpha in Crohn's disease.

OBJECTIVE: A global gene expression profile of non-inflamed colonic mucosal cells from patients with Crohn's disease (CD) and of colonic mucosal cells from controls was performed. MATERIAL AND METHODS: Tissue specimens from macroscopically non-inflamed descending colon were obtained colonoscopically from 33 CD patients and from 17 control subjects. All controls and 10 CD patients were medication-free at the time of colonoscopy. The Human Genome U133 Plus 2.0 GeneChip Array was used for gene profiling. Hybridization data were analysed with dChip software. Results were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Protein product expression of selected genes was assessed by immunohistochemistry using the Envision+ visualization technique. RESULTS: The expression profile was not homogeneous with the statistical cut-point settings applied. In comparison with controls, it was found that 19 CD patients had three differentially expressed genes, two of them related to the innate immune system: dual oxidase 2 on chromosome 15 (DUOX2, fold change 4.1) and lipocalin 2 on chromosome 9 (LCN2, fold change 3.1). The third gene, regenerating islet-derived 1 alpha (REG1A, fold change 3.9), codes for a mitogenic protein; this could not be confirmed by RT-PCR. Medication-free patients had no differentially expressed genes as compared with controls. Immunohistochemistry indicated that these proteins were produced by epithelial cells (REG1A, LCN2) and leucocytes (DUOX2 and LCN2). CONCLUSIONS: As compared with controls, non-inflamed colonic mucosal cells contain two up-regulated genes related to the innate immune system. Up-regulation of these genes, known to be induced by microorganisms, suggests either increased microflora antigenicity or an altered function in mucosal barrier defence.

Characterization of cecal gene expression in a differentially susceptible mouse model of bacterial-induced inflammatory bowel disease.

BACKGROUND: A/JCr mice develop typhlitis in response to Helicobacter hepaticus infection, whereas C57BL/6 mice coexist with this bacterium in a "commensal" relationship and do not develop disease even during prolonged colonization. METHODS: To determine mechanisms that control this balance between responsiveness and nonresponsiveness, the mucosal response of A/JCr and C57BL/6 mice to acute H. hepaticus colonization was evaluated using genome-wide profiling. Transcription levels for a subset of gene discoveries were then evaluated longitudinally by semiquantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) to identify changes in gene expression that occur during progression from the acute to chronic phase of colonization. To determine whether chronic mucosal inflammation in A/JCr mice was mediated through a Th1 mechanism, as was inferred from the gene expression data, mice with typhlitis were treated with neutralizing antibody targeting IL-12/23p40 or IFN-gamma and the response to treatment was determined by cecal lesion severity and transcription of disease-related genes. RESULTS: A/JCr mice had a biphasic expression of proinflammatory genes that corresponded with the acute and chronic phases of disease. In contrast, C57BL/6 mice exhibited a less robust acute transcriptional response that waned by day 30 postinoculation. Sustained upregulation of proinflammatory signals and responsiveness to anti-IL-12/23p40 and anti-IFN-gamma antibody suggests that inflammation in A/JCr mice was mediated through a Th1 mechanism. Prolonged upregulation of SOCS3 during the acute response to colonization suggests that C57BL/6 mice maintain mucosal homeostasis, at least in part by attenuating responsiveness to cytokine signaling. CONCLUSIONS: Collectively, these findings provide a foundation for understanding the immunological mechanisms that confer resistance or susceptibility to H. hepaticus-induced typhlitis.

Functional antagonism between RNA binding proteins HuR and CUGBP2 determines the fate of COX-2 mRNA translation.

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) expression is regulated at the levels of messenger RNA (mRNA) stability and translation by AU-rich elements (ARE) located in its 3' untranslated region (3'UTR). Although structurally homologous RNA binding proteins HuR and CUGBP2 stabilize COX-2 mRNA, HuR induces whereas CUGBP2 inhibits COX-2 mRNA translation. This study aimed to determine the antagonism between these proteins on COX-2 expression. METHODS: COX-2 ARE binding activity was determined by nitrocellulose filter binding and UV cross-linking assays using recombinant glutathione S-transferase (GST)/HuR and GST/CUGBP2. Protein:protein interactions were determined by GST pull-down, yeast 2-hybrid, and immunocytochemistry assays. Nucleocytoplasmic shutting was determined by heterokaryon analyses. The effect of CUGBP2 and HuR on COX-2 ARE-dependent translation was shown by a chimeric luciferase mRNA containing COX-2 3'UTR. HT-29 cells were subjected to 12 Gy gamma-irradiation in a cesium irradiator. RESULTS: CUGBP2 and HuR bind with similar affinities to COX-2 ARE, but CUGBP2 competes with HuR for binding. In vitro, HuR and CUGBP2 heterodimerize. Furthermore, FLAG-tagged HuR and myc-tagged CUGBP2 colocalize in the nucleus of HCT-116 cells. Moreover, both proteins shuttled between the nucleus and cytoplasm. In vitro, HuR enhanced whereas CUGBP2 inhibited translation of the chimeric luciferase COX-2 3'UTR mRNA. Furthermore, CUGBP2 competitively inhibited HuR-mediated translation of the transcript. In HT-29 cells transfected with HuR and CUGBP2, a switch in COX-2 mRNA binding from predominantly HuR to CUGBP2 occurred after radiation treatment, which was coupled with increased silencing of the COX-2 mRNA. CONCLUSIONS: CUGBP2 overrides HuR and suppresses COX-2 mRNA translation.

Dysregulation of Reg gene expression occurs early in gastrointestinal tumorigenesis and regulates anti-apoptotic genes.

Expression of anti-apoptotic genes is frequently elevated in tumors, where they increase resistance to chemotherapeutic agents and predict poor patient outcomes. However, key cellular factors regulating anti-apoptotic genes in tumors remain unknown. Increased expression of the regenerating (Reg) genes is commonly observed in gastrointestinal (GI) malignancies including colorectal cancer (CRC). We therefore examined Reg gene expression and associated changes in anti-apoptotic genes in an animal model of GI tumorigenesis. Using real time RT-PCR, we measured expression of Reg genes in human colorectal adenocarcinoma specimens, colon adenocarcinoma cell lines and adenomas from multiple intestinal neoplasia (min) mice heterozygous for a germ-line mutation of the adenomatous polyposis coli (APC) gene. Expression of Reg genes is increased in human colorectal adenocarcinomas and in the intestine of APCmin/+ mice at four weeks of age, a time preceding the spontaneous second mutation in the APC gene. Individual Reg genes exhibited regional expression profiles across the GI tract in mice. Adenomas from 14-week old mice had significant increases in at least one member of the Reg gene family, most commonly Reg IV and an associated increase in expression of the anti-apoptotic gene, Bcl-2. Addition of exogenous recombinant human Reg IV to human colon adenocarcinoma cells significantly increased Bcl-2 and Bcl-xL expression and induced resistance to ionizing radiation. These results show that dysregulation of Reg genes occur early in tumorigenesis. Furthermore, increased expression of Reg genes, specifically Reg IV contribute to adenoma formation and lead to increased resistance to apoptotic cell death in CRC.

Growth factors as treatment options for intestinal inflammation.

On the basis of several studies that have been completed to date, some growth factors appear promising for the treatment of inflammatory bowel disease: keratinocyte-like growth factor-2 (KGF-2), epidermal growth factor (EGF) enemas used in combination with oral mesalamine, somatropin (human growth hormone), and sargramostim (recombinant human GM-CSF). The results of these studies are highlighted and suggest that new insights into the regulation of intestinal immunity may provide effective synergistic or single-agent treatment alternatives to immunosuppression for inflammatory bowel disease. These data focus on the reparative components of mucosal homeostasis.