OLFM4 mRNA Research Articles

Profiling intestinal stem and proliferative cells in the small intestine of broiler chickens via in situ hybridization during the peri-hatch period

Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5, and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.

Identification of a glycolysis- and lactate-related gene signature for predicting prognosis, immune microenvironment, and drug candidates in colon adenocarcinoma.

Background: Colon adenocarcinoma (COAD), a malignant gastrointestinal tumor, has the characteristics of high mortality and poor prognosis. Even in the presence of oxygen, the Warburg effect, a major metabolic hallmark of almost all cancer cells, is characterized by increased glycolysis and lactate fermentation, which supports biosynthesis and provides energy to sustain tumor cell growth and proliferation. However, a thorough investigation into glycolysis- and lactate-related genes and their association with COAD prognosis, immune cell infiltration, and drug candidates is currently lacking. Methods: COAD patient data and glycolysis- and lactate-related genes were retrieved from The Cancer Genome Atlas (TCGA) and Gene Set Enrichment Analysis (GSEA) databases, respectively. After univariate Cox regression analysis, a nonnegative matrix factorization (NMF) algorithm was used to identify glycolysis- and lactate-related molecular subtypes. Least absolute shrinkage and selection operator (LASSO) Cox regression identified twelve glycolysis- and lactate-related genes (ADTRP, ALDOB, APOBEC1, ASCL2, CEACAM7, CLCA1, CTXN1, FLNA, NAT2, OLFM4, PTPRU, and SNCG) related to prognosis. The median risk score was employed to separate patients into high- and low-risk groups. The prognostic efficacy of the glycolysis- and lactate-related gene signature was assessed using Kaplan–Meier (KM) survival and receiver operating characteristic (ROC) curve analyses. The nomogram, calibration curves, decision curve analysis (DCA), and clinical impact curve (CIC) were employed to improve the clinical applicability of the prognostic signature. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on differentially expressed genes (DEGs) from the high- and low-risk groups. Using CIBERSORT, ESTIMATE, and single-sample GSEA (ssGSEA) algorithms, the quantities and types of tumor-infiltrating immune cells were assessed. The tumor mutational burden (TMB) and cytolytic (CYT) activity scores were calculated between the high- and low-risk groups. Potential small-molecule agents were identified using the Connectivity Map (cMap) database and validated by molecular docking. To verify key core gene expression levels, quantitative real-time polymerase chain reaction (qRT–PCR) assays were conducted. Results: We identified four distinct molecular subtypes of COAD. Cluster 2 had the best prognosis, and clusters 1 and 3 had poor prognoses. High-risk COAD patients exhibited considerably poorer overall survival (OS) than low-risk COAD patients. The nomogram precisely predicted patient OS, with acceptable discrimination and excellent calibration. GO and KEGG pathway enrichment analyses of DEGs revealed enrichment mainly in the “glycosaminoglycan binding,” “extracellular matrix,” “pancreatic secretion,” and “focal adhesion” pathways. Patients in the low-risk group exhibited a larger infiltration of memory CD4+ T cells and dendritic cells and a better prognosis than those in the high-risk group. The chemotherapeutic agent sensitivity of patients categorized by risk score varied significantly. We predicted six potential small-molecule agents binding to the core target of the glycolysis- and lactate-related gene signature. ALDOB and APOBEC1 mRNA expression was increased in COAD tissues, whereas CLCA1 and OLFM4 mRNA expression was increased in normal tissues. Conclusion: In summary, we identified molecular subtypes of COAD and developed a glycolysis- and lactate-related gene signature with significant prognostic value, which benefits COAD patients by informing more precise and effective treatment decisions.

Runting Stunting Syndrome in Broiler Chickens Is Associated with Altered Intestinal Stem Cell Morphology and Gene Expression.

Runting stunting syndrome (RSS) in broiler chickens is characterized by altered intestinal morphology and gene expression and stunted growth. The objective of this study was to conduct a retrospective study of gene expression in stem and differentiated cells in the small intestine of RSS chicks. Two different models of RSS were analyzed: broiler chicks that were experimentally infected and broiler chicks that were naturally infected. Experimentally infected chicks were exposed to litter from infected flocks (RSS-litter chicks) or infected with astrovirus (RSS-astrovirus chicks). Intestinal samples from naturally infected chicks showing clinical signs of RSS were acquired from commercial farms in Georgia and were brought into a poultry diagnostic lab (RSS-clinical-GA) and from farms in Brazil that had a history of RSS (RSS-clinical-BR). The RSS-clinical-BR chicks were separated into those that were positive or negative for gallivirus based on DNA sequencing. Intestinal morphology and intestinal cell type were identified in archived formalin-fixed, paraffin-embedded tissues. In situ hybridization for cell-specific mRNA was used to identify intestinal stem cells expressing olfactomedin 4 (Olfm4), proliferating cells expressing Ki67, absorptive cells expressing sodium glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), and goblet cells expressing mucin 2 (Muc2). RSS-litter and RSS-clinical-GA chicks showed 4% to 7.5% cystic crypts, while gallivirus-positive RSS-clinical-BR chicks showed 11.7% cystic crypts. RSS-astrovirus and gallivirus-negative RSS-clinical-BR chicks showed few cystic crypts. RSS-litter and gallivirus-positive RSS-clinical-BR chicks showed an increase in crypt depth compared to control or gallivirus-negative chicks, respectively. There was no expression of Olfm4 mRNA in the stem cells of RSS-litter and RSS-clinical-GA chicks, in contrast to the normal expression of Olfm4 mRNA in RSS-astrovirus and RSS-clinical-BR chicks. All chicks regardless of infection status showed normal expression of Ki67 mRNA in crypt cells, Muc2 mRNA in goblet cells, and SGLT1 or PepT1 mRNA in enterocytes. These results demonstrate that RSS, which can be induced by different etiologies, can show differences in the expression of the stem cell marker Olfm4.

Senescence-accelerated mouse prone 8 mice exhibit specific morphological changes in the small intestine during senescence and after pectin supplemented diet.

Impairment of gastrointestinal function and reduction of nutrient absorption associated with aging contribute to increased risk of malnutrition in the elderly population, resulting in physical weakness and vulnerability to disease. The present study was performed to examine the relationships between aging-associated morphological changes of the small intestine and nutrient malabsorption using senescence-accelerated mouse prone 8 (SAMP8) mice. Comparison of the morphology of the small intestine of young (22-week-old) and senescent (43-week-old) SAMP8 mice showed no significant changes in villus length, while the mRNA expression levels of secretory cell marker genes were significantly reduced in senescent mice. In addition, crypts recovered from the small intestine of senescent mice showed a good capacity to form intestinal organoids ex vivo, suggesting that the regenerative capacity of intestinal stem cells (ISCs) was unaffected by accelerated senescence. These results indicated that changes induced by accelerated senescence in the small intestine of SAMP8 mice are different from changes reported previously in normal aging mouse models. Biochemical analyses of serum before and during senescence also indicated that senescent SAMP8 mice are not in a malabsorption state. Furthermore, a diet supplemented with persimmon pectin had a mild effect on the small intestine of senescent SAMP8 mice. Intestinal villus length was slightly increased in the medial part of the small intestine of pectin-fed mice. In contrast, intestinal crypt formation capacity was enhanced by the pectin diet. Organoid culture derived from the small intestine of mice fed pectin exhibited a greater number of lobes per organoid compared with those from mice fed a control diet, and Lyz1 and Olfm4 mRNA levels were significantly increased. In conclusion, accelerated senescence induced exclusive changes in the small intestine, which were not related to nutrient malabsorption. Therefore, the SAMP8 strain may not be a suitable model to evaluate the effects of aging on intestinal homeostasis and nutrient absorption impairment.

Identification of cells expressing OLFM4 and LGR5 mRNA by in situ hybridization in the yolk sac and small intestine of embryonic and early post-hatch chicks

The chicken yolk sac (YS) and small intestine are essential for nutrient absorption during the pre-hatch and post-hatch periods, respectively. Absorptive enterocytes and secretory cells line the intestinal villi and originate from stem cells located in the intestinal crypts. Similarly, in the YS, there are absorptive and secretory cells that presumably originate from a stem cell population. Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5) and olfactomedin 4 (Olfm4) are 2 widely used markers for intestinal stem cells. The objective of this study was to map the distribution of putative stem cells expressing LGR5 and OLFM4 mRNA in the chicken small intestine from the late embryonic period to early post hatch and the YS during embryogenesis. At embryonic d 11, 13, 15, 17, and 19, the YS was collected (n = 3), and small intestine was collected at embryonic d 19, d of hatch (doh), and d 1, 4, and 7 post hatch (n = 3). Cells expressing OLFM4 and LGR5 mRNA were identified by in situ hybridization. In the YS, cells expressing only LGR5 and not OLFM4 mRNA were localized to the vascular endothelial cells lining the blood vessels. In the small intestine, cells in the intestinal crypt expressed both LGR5 and OLFM4 mRNA. Staining for OLFM4 mRNA was more intense than LGR5 mRNA, demonstrating that Olfm4 is a more robust marker for stem cells than Lgr5. At embryonic d 19 and doh, cells staining for OLFM4 mRNA were already present in the rudimentary crypts, with the greatest staining in the duodenal crypts. The intensity of OLFM4 mRNA staining increased from doh to d 7 post hatch. Dual label staining at doh for the peptide transporter PepT1 and Olfm4 revealed a population of cells above the crypts that did not express Olfm4 or PepT1 mRNA. These cells are likely progenitor transit amplifying cells. Thus, avians and mammals share similarity in the ontogeny of stem cells in the intestinal crypts.

Expression of Wnt and Notch signaling pathways in inflammatory bowel disease treated with mesenchymal stem cell transplantation: evaluation in a rat model.

IntroductionThe purpose of this study was to investigate the expression of Wnt and Notch signaling pathway-related genes in inflammatory bowel disease (IBD) treated with mesenchymal stem cell transplantation (MSCT).MethodsTNBS (2,4,6-trinitrobenzene sulfonic acid) was used to establish IBD in a rat model. Mesenchymal stem cells (MSCs) were transplanted via tail vein transfusion. Saline water was used in a control group. The expression of Wnt and Notch main signaling molecules was screened by gene chips and verified by quantitative reverse transcription-polymerase chain reaction in the IBD rat model on day 14 and day 28 after transplantation.ResultsThe IBD rat models were successfully established and MSCs were transplanted into those models. Genome-wide expression profile chips identified a total of 388 differentially expressive genes, of which 191 were upregulated and 197 were downregulated in the MSC-transplanted group in comparison with the IBD control group. Real-time quantitative polymerase chain reaction results showed that the level of Olfm4 mRNA expression in the IBD group (2.54±0.20) was significantly increased compared with the MSCT group (1.39±0.54) and the normal group (1.62±0.25) (P <0.05). The Wnt3a mRNA was more highly expressed in IBD rats (2.92±0.94) and decreased in MSCT rats (0.17±0.63, P <0.05). The expression of GSK-3β mRNA was decreased in the setting of inflammation (0.65±0.04 versus 1.00±0.01 in normal group, P <0.05) but returned to normal levels after MSCT (0.81±0.17). The expression of β-catenin was observed to increase in IBD tissues (1.76±0.44) compared with normal tissues (1.00±0.01, P <0.05), but no difference was found in the MSCT group (1.12±0.36). Wnt11 declined at 14 days and returned to normal levels at 28 days in the IBD group; in comparison, a significantly lower expression was found in MSCT rats. There were no differences in the expression of Fzd3, c-myc, TCF4, and Wnt5a in inflammation, but all of those genes declined after MSCT treatment.ConclusionsThe canonical Wnt and Notch signaling pathways are activated in IBD and may be suppressed by stem cell transplantation to differentiate into intestinal epithelium after MSCT. Moreover, the non-canonical Wnt signaling may be inhibited by canonical Wnt signaling in the setting of inflammation and may also be suppressed by MSCT.

A Lack of Secretory Leukocyte Protease Inhibitor (SLPI) Causes Defects in Myeloid Differentiation

AbstractAbstract 10Previously, we demonstrated severe diminished mRNA and protein levels of secretory leukocyte protease inhibitor (SLPI) in CD33+ myeloid cells in PMNs and in plasma of patients with severe congenital neutropenia (CN), as compared to G-CSF treated healthy individuals. We further analysed whether diminished levels of SLPI are associated with the “maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI in the CD34+ bone marrow hematopoietic progenitor cells and in the acute myelid leukemia cell line NB4 using lentivirus-based transduction with SLPI-specific shRNA and analysed G-CSF- or ATRA-triggered myeloid differentiation, respectively. We found that G-CSF-triggered myeloid differentiation of CD34+ cells transduced with SLPI-specific shRNA was significantly diminished, in comparison to ctrl shRNA transduced cells (on day 14 of differentiation 53.8 % of CD11b+ cells in SLPI shRNA group vs 86.3 % in the ctrl shRNA group (ttest: p = 0.005); 13.1 % of CD16+ cells in SLPI shRNA group vs 48.8 % in the ctrl shRNA group (ttest: p = 0.0006)). This was accompanied by G0/G1 cell cycle arrest and elevated apoptosis of SLPI shRNA transduced cells: on day 7 of differentiation 83.9 % of SLPI shRNA transduced cells and 66.6 % of the ctrl shRNA transduced cells were in G0/G1 phase (ttest: p = 0.03); 11 % of annexin V positive cells in SLPI shRNA transduced group vs 6.5 % in ctrl shRNA group (ttest: p = 0.004). Similar effects on the ATRA-induced myeloid differentiation were observed after inhibition of SLPI in the NB4 cell line.We next analysed the mechanisms of diminished myeloid differentiation due to diminished SLPI levels. SLPI is a cationic serine protease inhibitor with antiprotease, primarily anti-neutrophil elastase, activities. Previously we showed regulation of neutrophil elastase by SLPI in myeloid cells. However, SLPI is also known to modulate intracellular signal transduction pathways such as NF-kB and Erk. SLPI is involved in the phosphorylation of Erk1/2 protein. Erk1/2 protein plays a role in myeloid differentiation. We found that ATRA treatment of NB4 cells transduced with ctrl shRNA led to the transient time-dependent phosphorylation of Erk1/2 on day 2 of culture with subsequent decline on day 4 of treatment. Interestingly, Erk1/2 phosphorylation was almost completely abolished on day 2 of ATRA treatment of NB4 cells transduced with SLPI shRNA. Recently, we demonstrated an essential role of LEF-1 in the granulocytic differentiation and severe diminished LEF-1 expression levels in myeloid cells of CN patients. It has been shown that Erk1/2 phosphorylates LEF-1 transcription factor. In line with defective phosphorylation of Erk1/2 we measured severe diminished levels of phosphorylated LEF-1 in SLPI shRNA transduced cells, in comparison to ctrl shRNA samples. Therefore, we concluded that SLPI regulates myeloid differentiation by regulation of Erk1/2 dependent LEF-1 phosphorylation and autoregulation.To evaluate additional intracellular signaling pathways which are affected during G-CSF-triggered myeloid differentiation if SLPI is downregulated, we performed microarray analysis of RNA from CD34+ cells transduced with SLPI or ctrl shRNA and subsequently treated with G-CSF for 24 hours. Analysis of the microarray data using Ingenuity Systems Pathway Analysis (IPA) revealed severe significant defects in the signaling cascades involved in the differentiation (p-Value = 0.0093), proliferation (p-Value = 0.015), survival (p-Value = 0.012) and chemotaxis (p-Value = 0.0093) of myeloid progenitor cells and neutrophils which was correlated with significant downregulation of FPR2, MMP8, XCL1, S100A9, IL2 and OLFM4 mRNA expression. Moreover, signaling systems regulating DNA replication checkpoint and arrest in Go/G1 phase were also significantly affected (p-Value = 0.0009 and 0.0065, respectively) due to downregulation of mRNA expression of CHEK1, CCNE2, CDC6, MYBL1 and MYC.Taken together, SLPI is a new candidate factor playing an important role in myelopoiesis by controlling proliferation, differentiation and cell cycle of myeloid cells not only by inhibition of neutrophil elastase but also by regulation of intracellular signaling pathways. Disclosures:No relevant conflicts of interest to declare.

302 Dynamic Regulation of Intestinal Crypt Base Columnar Stem Cells by Notch Signaling

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

303 High Altitude Journeys and Flights are Associated With the Increased Risk of Flares in IBD Patients

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

Olfactomedin 4 defines a subset of human neutrophils

OLFM4 was identified initially as a gene highly induced in myeloid stem cells by G-CSF treatment. A bioinformatics method using a global meta-analysis of microarray data predicted that OLFM4 would be associated with specific granules in human neutrophils. Subcellular fractionation of peripheral blood neutrophils demonstrated complete colocalization of OLFM4 with the specific granule protein NGAL, and stimulation of neutrophils with PMA resulted in corelease of NGAL and OLFM4, proving that OLFM4 is a genuine constituent of neutrophil-specific granules. In accordance with this, OLFM4 mRNA peaked at the MY/MM stage of maturation. OLFM4 was, however, present in only 20-25% of peripheral blood neutrophils, as determined by immunocytochemistry and flow cytometry, whereas mRNA for OLFM4 was present in all MY/MM, indicating post-transcriptional regulation as a basis for the heterogeneous expression of OLFM4 protein.

Olfactomedin-4 is a glycoprotein secreted into mucus in active IBD

BackgroundOlfactomedin-4 (OLFM4) is a glycoprotein characteristic of intestinal stem cells and apparently involved in mucosal defense of the stomach and colon. Here we studied its expression, regulation and function in IBD. MethodsThe expression of OLFM4, mucins Muc1 and Muc2, the goblet cell differentiation factor Hath1 and the proinflammatory cytokine IL-8 was measured in inflamed or noninflamed colon in IBD patients and controls. OLFM4 protein was located by immunohistochemistry, quantified by Dot Blot and its binding capacity to defensins HBD1-3 was investigated. The influence of bacteria with or without the Notch blocker dibenzazepine (DBZ) and of several cytokines on OLFM4 expression was determined in LS174T cells. ResultsOLFM4 mRNA and protein were significantly upregulated in inflamed CD (4.3 and 1.7-fold) and even more pronounced in UC (24.8 and 3.7-fold). OLFM4 expression was correlated to IL-8 but not to Hath1. In controls immunostaining was restricted to the lower crypts but in inflamed IBD it expanded up to the epithelial surface including the mucus. OLFM4 bound to HBD1-3 without profoundly inactivating these defensins. In LS174T-cells OLFM4 mRNA was significantly augmented after incubation with Escherichia coli K12, Escherichia coli Nissle and Bacteroides vulgatus. DBZ downregulated OLFM4 expression and blocked bacterial induction whereas IL-22 but not TNF-α was stimulatory. ConclusionsOLFM4 is overexpressed in active IBD and secreted into mucus. The induction is triggered by bacteria through the Notch pathway and also by the cytokine IL-22. OLFM4 seems to be of functional relevance in IBD as a mucus component, possibly by binding defensins.

Notch-Hes1 Pathway and TNF-α Synergistically up-Regulates OLFM4 Expression in the Inflamed Mucosa of the Human Intestine

Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-αmediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific genemight be required to promote the regenerative response of the inflamed intestinal environment.

Slco1b1*1b Haplotype is Associated With Risk of Aspirin Induced Peptic Ulcer and Bleeding

Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-αmediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific genemight be required to promote the regenerative response of the inflamed intestinal environment.

A Novel Role for IL-27 as a Mediator of Intestinal Epithelial Barrier Protection Mediated via Differential Stat Signaling and Induction of Antibacterial and Anti-Inflammatory Proteins

Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-αmediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific genemight be required to promote the regenerative response of the inflamed intestinal environment.

Abstract 2168: Detections of olfactomedin 4 gene expression by qRT-PCR in human prostatic samples and cell lines

Abstract The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein. OLFM4 gene is normally expressed in a limited number of tissues, including the prostate, but the regulation of OLFM4 gene expression in prostate are largely unknown. We have previously reported that reduced or lost expression of OLFM4 protein is correlated with higher Gleason scores in prostate cancer and loss of gene expression is due to deletions within the OLFM4 gene in about 36.7% (11/30) of prostate cancer patient samples studied. In this study, we investigated the regulation of OLFM4 gene expression in human prostate cancer pathogenesis. We have investigated the mRNA expression levels using quantitative RT-PCR analysis in 144 human prostate cDNA samples (OriGene). We found that the average expression level of OLFM4 gene is significantly reduced in cancer tissues compared with normal tissues. The expression level of OLFM4 mRNA is inversely corrected with higher Gleason Score in prostate cancer. Interestingly, we also found higher expression of OLFM4 gene within small subset of samples with inflammation including prostatitis, hyperplasia and well differentiated tumor tissue. We have also screened the expression of OLFM4 mRNA level using qRT-PCR in primary cultured human normal prostate epithelial cells (PREC), benign immortalized prostate epithelial cell (RWPE1), androgen-responsible cancer cells (LNCaP and VCaP), androgen-independent cancer cells (PC-3 and DU145). We found lower expression of OLFM4 in RWPE1 cells. We also confirmed our previous results that OLFM4 mRNA was not expressed in cancer cell lines, LNCaP, VCaP, PC-3 and DU145. We have reported previously that deletions within the OLFM4 gene were present in about 40% in human prostate cancer samples studied. To test whether methylation is also involved in the loss of OLFM4 gene expression in human prostate cancer, the methylation status of CpG sites in OLFM4 promoter region has been analyzed in primary prostate tissues obtained by Laser Capture Microdissection. Hypermethylation of CpG sites at -91, -486 and -681 were detected in higher Gleason score of cancer samples. We further treated PC-3 cells (in which the OLFM4 gene is intact) using the demethylation reagent, 5-aza-2’-deoxycytidine and found restoration of OLFM4 mRNA expression. The results suggest that methylation status of OLFM4 gene promoter region may affect OLFM4 gene expression in prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2168. doi:10.1158/1538-7445.AM2011-2168

Olfactomedin 4, An Indicator of the Existence of Neutrophil Subsets.

AbstractAbstract 3775Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules. [Display omitted] Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not. [Display omitted] Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist. Disclosures:No relevant conflicts of interest to declare.

Olfactomedin 4 promotes S‐phase transition in proliferation of pancreatic cancer cells

Induction of olfactomedin 4 (OLFM4(GW112/hGC-1)) in cancer cells was recently reported to have a novel antiapoptotic action via binding to the potent apoptosis inducer GRIM-19. We sought to clarify undiscovered functions of constitutively expressed OLFM4 in cancer cells. OLFM4 mRNA was highly expressed in pancreatic cancer tissues. In PANC-1 cell cultures, expression was especially elevated during early S phase of the cell cycle. Transduction of small interfering RNA for OLFM4 to decrease mRNA expression caused time-dependent growth inhibition, with typical early S-phase arrest after 6 days. In addition, cell volume increased without increases in multinucleated cells, consistent with premitotic inhibition of DNA synthesis. Inhibition of OLFM4 mRNA expression by small interfering RNA did not promote apoptosis. Taken together, the results indicate that OLFM4 promotes proliferation of PANC-1 cells by favoring transition from the S to G(2)/M phase.

Specific overexpression of OLFM4GW112/hGC−1 mRNA in colon, breast and lung cancer tissues detected using quantitative analysis

Overexpression of the olfactomedin 4 (OLFM4(GW112,/hGC-1)) gene was recently reported to inhibit various apoptotic pathways and promote proliferation of cancer cells, suggesting that OLFM4 might serve as a diagnostic marker for human cancers. Therefore, we examined cancer-specific OLFM4 overexpression. OLFM4 mRNA was highly expressed in cancerous tissues obtained from the colon, breast and lung. Positivity for OLFM4 mRNA, defined as the mean + 2 SD in non-cancerous colon and breast tissues, was observed in 68 and 50% of the studied colon and breast cancer tissues. OLFM4 mRNA expression was not detected in non-cancerous lung tissues but was evident in 62% of the lung cancer tissues. On comparing paired samples, the expression of OLFM4 mRNA was observed to be elevated in 90, 69 and 85% of colon, breast and lung cancer tissues, respectively. OLFM4 mRNA expression was observed even in the early stages of each cancer type. The expression of OLFM4 mRNA did not correlate with that of the antiapoptotic molecule survivin, indicating that it can be used independently in cancer diagnosis. Combining OLFM4 and survivin resulted in higher positivity. Thus, OLFM4 mRNA might be a useful tool to support the diagnosis of cancer, irrespective of the clinical stages.