The Jagged-1/Notch-1/Hes-1 Pathway Is Involved in Intestinal Adaptation in a Massive Small Bowel Resection Rat Model

Background

Notch signaling is required for the maintenance of intestinal epithelial proliferation. Dysfunction of this signaling pathway is associated with the loss of proliferated crypt epithelial cells.

Aim

The aim of this study was to investigate the role of Notch signaling in small bowel resection (SBR)-associated crypt epithelial cell proliferation.

Methods

Male Sprague–Dawley rats were subjected to sham operation (bowel transection and reanastomosis) or 70 % mid-SBR. Intestinal tissue samples were collected at 0.5, 1, 6, 12, 24, 72, and 168 h after operation. The expression of Notch pathway mRNAs and proteins was analyzed using RT-PCR and Western blot. The expression of the Notch pathway proteins Jagged-1, NICD and Hes-1 was also determined through immunohistochemical staining using day 3 postoperative intestinal tissues. The degree of crypt epithelial cell proliferation was evaluated using the immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Furthermore, IEC-6 cells were used to examine the function of the Jagged-1 signaling system.

Results

SBR led to increased crypt epithelial cell proliferation and increased expression of Jagged-1 and Hes-1 mRNA and protein along with cleaved Notch-1. Immunohistochemical staining showed that Jagged-1, cleaved Notch-1 and Hes-1 colocalized in the same proliferated crypt epithelial cell population. Recombinant Jagged-1 significantly stimulated the proliferation of IEC-6 cells. Transient upregulation of Jagged-2 expression was found 1 h after SBR, and it was accompanied by cleaved Notch-1 and Hes-1 upregulation.

Conclusion

The Jagged-1/Notch-1/Hes-1 signaling pathway is involved in intestinal adaptation through increasing crypt epithelial cell proliferation.     

Expression and localization of axin 2 in colorectal carcinoma and its clinical implication

Purpose

Aberrant activation of the Wnt/β-catenin pathway plays a major role in the development of colorectal carcinoma (CRC). Axin 2 is a key protein of this pathway and is upregulated in CRC. Here, we investigated RNA- and protein expression of axin 2 in CRC tissues at the single cell level. Moreover, the association of axin 2 with prognosis and survival was investigated in a large cohort of CRC patients (n?=?280).

Methods

Localization and expression of axin 2 and β-catenin was investigated using in situ hybridization and immunohistochemical staining. The quantitative expression levels of axin 2 were determined using RT-qPCR. The association of axin 2 expression with prognosis and survival of the patients was determined by statistical analysis (logrank test, Kaplan–Meier).

Results

Our results confirmed the upregulation of axin 2 in CRC and showed that it is broadly expressed in the cytoplasm of the tumor epithelial cells both, in the tumor center and at the invasion front. Axin 2 was rarely expressed by tumor stromal cells and only weakly by normal colonic epithelial cells. Staining of β-catenin and axin 2 in consecutive CRC tissue sections revealed that nuclear translocation of β-catenin in the tumor front was not associated with changes in the cytoplasmic localization of axin 2. Axin 2 did not show any association with proven prognostic factors or survival of the CRC patients.

Conclusion

The generally increased expression of axin 2 in all tumor stages as compared to normal tissue suggests an initiating pathogenic function in the development of CRC.     

Wnt-reporter expression pattern in the mouse intestine during homeostasis

Background

The canonical Wnt signaling pathway is a known regulator of cell proliferation during development and maintenance of the intestinal epithelium. Perturbations in this pathway lead to aberrant epithelial proliferation and intestinal cancer. In the mature intestine, proliferation is confined to the relatively quiescent stem cells and the rapidly cycling transient-amplifying cells in the intestinal crypts. Although the Wnt signal is believed to regulate all proliferating intestinal cells, surprisingly, this has not been thoroughly demonstrated. This important determination has implications on intestinal function, especially during epithelial expansion and regeneration, and warrants an extensive characterization of Wnt-activated cells.

Methods

To identify intestinal epithelial cells that actively receive a Wnt signal, we analyzed intestinal Wnt-reporter expression patterns in two different mouse lines using immunohistochemistry, enzymatic activity, in situ hybridization and qRT-PCR, then corroborated results with reporter-independent analyses. Wnt-receiving cells were further characterized for co-expression of proliferation markers, putative stem cell markers and cellular differentiation markers using an immunohistochemical approach. Finally, to demonstrate that Wnt-reporter mice have utility in detecting perturbations in intestinal Wnt signaling, the reporter response to gamma-irradiation was examined.

Results

Wnt-activated cells were primarily restricted to the base of the small intestinal and colonic crypts, and were highest in numbers in the proximal small intestine, decreasing in frequency in a gradient toward the large intestine. Interestingly, the majority of the Wnt-reporter-expressing cells did not overlap with the transient-amplifying cell population. Further, while Wnt-activated cells expressed the putative stem cell marker Musashi-1, they did not co-express DCAMKL-1 or cell differentiation markers. Finally, gamma-irradiation stimulated an increase in Wnt-activated intestinal crypt cells.

Conclusion

We show, for the first time, detailed characterization of the intestine from Wnt-reporter mice. Further, our data show that the majority of Wnt-receiving cells reside in the stem cell niche of the crypt base and do not extend into the proliferative transient-amplifying cell population. We also show that the Wnt-reporter mice can be used to detect changes in intestinal epithelial Wnt signaling upon physiologic injury. Our findings have an important impact on understanding the regulation of the intestinal stem cell hierarchy during homeostasis and in disease states.     

Identification of an intracellular metabolic signature impairing beta cell function in the rat beta cell line INS-1E and human islets

Aims/hypothesis

Chronic hyperglycaemia promotes the progressive failure of pancreatic beta cells in patients with type 2 diabetes mellitus, a clinically highly relevant phenomenon known as glucotoxicity. The intracellular metabolic consequences of a chronically high availability of glucose in beta cells are, as yet, poorly understood in its full complexity.

Methods

An unbiased metabolite profiling analysis (GC-time-of-flight-MS) was used to identify the time course of core metabolite patterns in rat beta cell line INS-1E during exposure to high glucose concentrations and its relation to insulin expression.

Results

We report here that pentose phosphate pathway (PPP) metabolites accumulate remarkably during chronic but not acute glucose treatment, indicating altered processing of glucose through the pentose phosphate pathway. Subsequent functional studies in INS-1E cells and human islets revealed that a disturbance in this pathway contributes to decreases in insulin gene expression and a lack of glucose-stimulated insulin secretion. These effects were found to depend on the activation of extracellular-regulated-kinase (ERK1/2). Long-term inhibition of 6-phosphogluconic acid dehydrogenase resulted in accumulation of PPP metabolites, induced ERK1/2 activation independently of high glucose and impaired beta cell function. In turn, inhibition of ERK1/2 overstimulation during chronic glucose exposure partly inhibited metabolite accumulation and restored beta cell function.

Conclusions/interpretation

Based on unbiased metabolite analyses, the data presented here provide novel targets, namely the inhibition of PPP metabolite accumulation towards the therapeutic goal to preserve and potentially improve beta cell function in diabetes.     

Matrix metalloproteinase 12–dependent cleavage of urokinase receptor in systemic sclerosis microvascular endothelial cells results in impaired angiogenesis

Objective

Defective angiogenesis, resulting in tissue ischemia, is particularly prominent in the diffuse form of systemic sclerosis (SSc). The present study was undertaken to identify possible differences between normal and SSc microvascular endothelial cells (MVECs) in the expression of the cell‐associated urokinase‐type plasminogen activator (uPA)/uPA receptor (uPAR) system, which is critical in the angiogenic process.

Methods

MVECs were isolated from the dermis of healthy individuals and from the dermis of patients with diffuse SSc. The uPA/uPAR system was examined at the protein and messenger RNA levels. Angiogenesis was assayed on Matrigel‐coated porous filters and plates to evaluate cell proliferation, invasion, and capillary morphogenesis. Cleavage of uPAR and the activity of matrix metalloproteinase 12 (MMP‐12) were evaluated by Western blotting.

Results

Compared with MVECs from healthy skin, MVECs from SSc patients showed higher expression of uPAR. However, in SSc MVECs, uPAR undergoes truncation between domain 1 and domain 2, as shown by flow cytometry, enzyme‐linked immunosorbent assay, and Western blotting, a cleavage that is known to impair uPAR functions. These properties of SSc MVECs were associated with poor spontaneous and uPA‐dependent invasion, proliferation, and capillary morphogenesis. The uPAR cleavage occurring in SSc MVECs was associated with overexpression of MMP‐12. SSc MVEC–conditioned medium impaired uPA‐dependent proliferation and invasion as well as capillary morphogenesis in normal MVECs in vitro. Both a general hydroxamate inhibitor of MMP activity and anti–MMP‐12 antibodies restored this SSc MVEC–induced impaired functioning.

Conclusion

Overproduction of MMP‐12 by SSc MVECs accounts for the cleavage of uPAR and the impairment of angiogenesis in vitro and may contribute to reduced angiogenesis in SSc patients.

     

Heat Shock Protein 27 Expression is Inversely Correlated with Atrophic Gastritis and Intraepithelial Neoplasia

Background

Intestinal-type gastric carcinomas progress through several sequential steps, including atrophic gastritis, intestinal metaplasia, dysplasia, and cancer.

Aim

We investigated heat shock protein 27 (HSP27) expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis.

Methods

We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and background gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues.

Results

HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of neoplasia-bearing patients was significantly lower than in the mucosa of those without gastric neoplasia. In tumor necrosis factor α-treated gastric cancer cells, HSP27 knockdown increased cell death and accumulation of the reactive oxygen species that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiation of cancer cells is associated with an epithelial–mesenchymal transition (EMT) signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells.

Conclusion

HSP27 expression in gastric mucosae is inversely correlated with intraepithelial neoplasia, a probable precursor to gastric cancer, and HSP27 expression in cancer is positively correlated with poor differentiation.     

Increased Gap Density Predicts Weakness of the Epithelial Barrier In Vivo by Confocal Laser Endomicroscopy in Indomethacin-Induced Enteropathy

Background and Aims

The intestinal epithelial barrier plays an important role in the pathogenesis of non-steroidal anti-inflammatory drug-induced enteropathy, and its disruption is often associated with increased cell shedding. The purpose of this report is to observe the gap density in indomethacin-induced small intestinal damage by confocal laser endomicroscopy (CLE) and to investigate the mechanisms involved in this process and how mucosal protectants improve intestinal epithelial barrier dysfunction. CLE is expected to provide a new way for evaluating non-steroidal anti-inflammatory drugs-induced enteropathy in humans and assessing drug efficacy.

Methods

Using the new technique of CLE, we established a method to evaluate, in real time, intestinal damage after the administration of indomethacin in Wistar rats by investigating the gap density in the small intestine. The mucosal protectant teprenone and acid-suppressant rabeprazole were then given by gavage before and after the administration of indomethacin, and the mechanisms affecting the intestinal epithelial barrier were investigated.

Results

Using CLE, gaps could be clearly observed and easily distinguished from goblet cells. Gap density was increased after the administration of indomethacin. During this process, the expression of tumor necrosis factor-α, nuclear factor-κB, and caspase-3 was up-regulated and the expression of tight junctions was down-regulated, which led to the damage of the epithelial barrier. Teprenone and rabeprazole could intervene in this pathway and protect the integrity of the epithelial barrier.

Conclusions

CLE can be objective, accurate, and real time in investigating gap density. Teprenone and rabeprazole can prevent indomethacin-induced intestinal lesions and protect the epithelial barrier by intervening in the tumor necrosis factor-α pathway. Gap density was expected to be an indicator of evaluating intestinal inflammation and drug efficacy.     

Detection of free fatty acid receptor 1 expression: the critical role of negative and positive controls

Aims/hypothesis

Adequate evaluation of protein expression is a crucial prerequisite for functional studies. Commonly used strategies comprise detection of proteins by specific antibodies using western blotting and immunohistochemical staining, or detection of mRNA by in situ hybridisation and RT-PCR. We evaluated the tools for the detection of free fatty acid receptor 1 (FFAR1) expression.

Methods

Commercially available antibody preparations were used to detect endogenous expression of the FFAR1 receptor and this was compared with cell preparations deficient or overexpressing the mouse or human receptor. Concentrations of mRNA were evaluated by RT-PCR.

Results

All insulin-secreting cells, INS-1E, Min6 and mouse islets showed specific expression of Ffar1 at the mRNA level. However, none of the commercially available antibodies specifically detected rat, mouse or human FFAR1.

Conclusions/interpretation

Proper positive and negative controls are an important prerequisite for the evaluation of FFAR1 expression.     

Effect of Cigarette Smoke Extract on Insulin Transport in Alveolar Epithelial Cell Line A549

Background

The main purpose of this study was to evaluate the effect of cigarette smoke extract (CSE) on insulin transport in alveolar epithelial cells.

Methods

We first examined the effect of CSE pretreatment on cell viability, mRNA expression, and lamellar body structures in A549 cells. Then the effect of CSE pretreatment on FITC-insulin transport was examined.

Results

When A549 cells were treated with 30???g/ml of CSE for 48?h, the expression of some mRNAs abundantly expressed in type II alveolar epithelial cells such as surfactant protein B was significantly increased. Lamellar bodylike structures became more evident with CSE treatment. FITC-insulin uptake from the apical side and subsequent efflux to the basal side was enhanced by CSE treatment in A549 cells. The enhancing effect of CSE on FITC-insulin uptake was concentration-dependent and reversible. A concentration-dependent enhancing effect of CSE on FITC-insulin uptake was also observed in normal, primary cultured alveolar type II epithelial cells isolated from rats.

Conclusions

Treatment of A549 cells by CSE may direct the cells to a more type II-like phenotype. In accordance with this observation, FITC-insulin uptake was enhanced by CSE treatment. These results may partly explain the higher insulin absorption from the lung in smokers than in nonsmokers.     

Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and uPA via MAPK pathways

Purpose

Sphingosine kinase (SphK) 1 is an oncogenic enzyme promoting transformation, proliferation, and survival of a number of human tumor cells. However, its effect on colon cancer cell behavior has not been fully clarified.

Methods

SphK1 plasmid or SphK1 shRNA transfection and N,N-dimethylsphingosine (DMS) was used to regulate the expression and activity of SphK1 in colon cancer line LOVO. Cell proliferation, apoptosis, invasion, and protein expression were detected by MTT, flow cytometry, transwell chambers model, and western blot. The levels of metalloproteinases-2/9 (MMP-2/9) and urokinase plasminogen activator (uPA) were detected by ELISA.

Results

Overexpression of SphK1 after plasmid transfection markedly enhanced LOVO cell viability and invasiveness and reduced cell apoptosis. In contrast, inhibition of SphK1 by DMS and shRNA significantly suppressed cell viability and invasiveness but promoted cell apoptosis. SphK1 increased the constitutive expression of extracellular signal-regulated kinase1/2 (ERK1/2) but reduced the constitutive expression of p38 mitogen-activated protein kinase (MAPK). Blocking ERK1/2 pathway inhibited the biological effects induced by overexpression of SphK1. Blocking p38 MAPK pathway reversed the effects of DMS and SphK1 shRNA. Moreover, SphK1 was required for the production of MMP-2/9 and uPA in tumor cells, which was suppressed by ERK1/2 inhibitor U0126, but enhanced by the p38 MAPK inhibitor SB203580.

Conclusions

SphK1 enhances colon cancer cell proliferation and invasiveness, meanwhile suppressing cell apoptosis. SphK1 promoting the secretion of MMP-2/9 and uPA via activation of ERK1/2 and suppression of p38 MAPK pathways maybe the molecular mechanisms for its regulation of the malignant behavior of colon cancer cell.     

SULF1 Inhibits Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells by Decreasing Heparin-Binding Growth Factor Signaling

Background

Heparin-binding growth factor signaling is involved in the pathogenesis and development of human cancers. It can be regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPG). SULF1 is a heparin-degrading endosulfatase which can modulate the sulfation of HSPGs.

Aim

The purpose of this study was to elucidate the role of SULF1 in modulating proliferation and invasion of esophageal squamous cell carcinoma (ESCC) by decreasing heparin-binding growth factor signaling.

Methods

We restored SULF1 expression in the ESCC cell line KYSE150, and examined the effects of SULF1 expression on the proliferation and invasion of KYSE150 cells. In addition, we investigated the expression of SULF1 in human ESCC tissues and analyzed the correlation of SULF1 expression with clinicopathologic characteristics of ESCC.

Results

Our study shows that re-expression of SULF1 in ESCC cell line results in the downregulation of hepatocyte growth factor-mediated activation of MAPK pathways with a resultant decrease in cell invasiveness. Cell proliferation was also inhibited in SULF1-transfected KYSE150 cells. Immunohistochemical assays reveal that SULF1 is expressed in nearly half of the human ESCC tissues but not in normal esophageal epithelial cells. SULF1 expression in human ESCC tissues is negatively correlated with tumor size and tumor invasion.

Conclusion

This study identified that SULF1 inhibits proliferation and invasion of ESCC by decreasing heparin-binding growth factor signaling and suggested that SULF1 plays an inhibiting role in the pathogenesis of ESCC.     

B and T lymphocyte attenuator is highly expressed on intrahepatic T cells during chronic HBV infection and regulates their function

Background

T cell antiviral function is impaired during chronic hepatitis B (CHB). Programmed death-1 (PD-1) impairs antiviral T cell responses, but dysfunction is not always reversed by blockade of PD-1 pathway. Whether distinct T cell populations expressing different sets of inhibitory molecules exist has not been determined.

Methods

We studied the expression of the B and T lymphocyte attenuator (BTLA) on both peripheral blood mononuclear cells (PBMC) and intrahepatic lymphocytes, and the effects of blocking BTLA on circulating and intrahepatic T cells in CHB patients. Sixty-three CHB patients who underwent liver biopsy were enrolled. The expression of BTLA and PD-1 on PBMC and intrahepatic T cells was assessed by flow cytometry with antibodies to T cell differentiation molecules. Functional recovery was evaluated by analyzing production of interferon (IFN)-γ and interleukin (IL)-2 after incubation of T cells with anti-CD3 and irradiated mature dendritic cells in the presence of anti-BTLA, anti-PD-1, or both.

Results

Intrahepatic T cells expressed higher levels of BTLA than their peripheral counterparts. A significant fraction of intrahepatic T cells coexpressed BTLA and PD-1 and showed deep exhaustion of T cell responses. Blockade of the BTLA pathway enhanced both intrahepatic and PBMC T cell proliferation and cytokine secretion, and exhibited an additive effect upon blockage of PD-1.

Conclusions

Upregulation of inhibitory receptor BTLA restricts T cell responses in CHB. T cell exhaustion by high antigen concentrations exacerbates dysfunction of peripheral and intrahepatic T cells. Blockage of BTLA is a potential therapeutic approach for chronic HBV infection that may act by restoring antiviral T cell responses.