Cytoprotective effect of epidermal growth factor on acid- and pepsininduced damage to rat gastric epithelial cells: Roles of Na+/H+ exchangers

We have previously established a cell damage model, with damage induced by either acid or pepsin treatment for 30 min, involving a rat gastric epithelial cell line (RGM1). In the present study, pretreatment of cells with epidermal growth factor (EGF; 0.1–10ng/mL) or sucralfate (0.1–3 mg/mL) for 4 h prevented such cell damage in a concentration-dependent manner. Protection of cells by these drugs was not affected by pretreatment with indomethacin (10⁻⁵ mol/L) for 4 h. Removal of Na⁻, but not Ca²⁺, from the acidified medium totally abolished the inhibitory effect of EGF, but not that of sucralfate. Genistein (a tyrosine kinase inhibitor) apparently reduced the inhibitory effect of EGF. DNA synthesis by RGM1 cells did not increase when cells were incubated with EGF for 4 h. We conclude that both EGF and sucralfate protect RGM1 cells from acid- and pepsin-induced damage and that the mechanism of protection by EGF against acid-induced damage seems to be via activation of Na⁺/H⁺ exchangers.     

Geranylgeranylacetone Induces Cyclooxygenase-2 Expression in Cultured Rat Gastric Epithelial Cells Through NF-κB

Geranylgeranylacetone (GGA) effectively protects the gastric mucosa against noxious agents. The precise mechanisms underlying the gastroprotective actions of GGA are not known. To elucidate the precise mechanism of GGA, the effect of GGA treatment on COX-2 expression in rat gastric epithelial (RGM1) cells was investigated. We used a prostaglandin E₂ (PGE₂) enzyme-linked immunoassay kit and Western blot analysis to measure PGE₂ production and COX-2 induction by GGA treatment in serum-starved RGM1 cells. Gel-shift assay, Western blot analysis, and a reporter assay were performed to determine which COX-2 promoter was involved in GGA-induced COX-2 expression. GGA treatment dose dependently increased COX-2 expression and PGE₂ production. The nuclear factor (NF)-κB sites of the COX-2 gene promoter were critical for GGA-mediated COX-2 expression. GGA induces COX-2 expression and increases PGE₂ production in serum-starved RGM1 cells via activation of the NF-κB sites of COX-2 gene promoters. The first two authors contributed equally to this work.     

Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey

The sea lamprey recovers normal-appearing locomotion after spinal cord transection and its spinal axons regenerate selectively in their correct paths. However, among identified reticulospinal neurons some are consistently bad regenerators and only about 50% of severed reticulospinal axons regenerate through the site of injury. We previously suggested (Shifman, M. I., and Selzer, M. E., 2000a. Expression of netrin receptor UNC-5 in lamprey brain; modulation by spinal cord transection. Neurorehabilitation and Neural Repair 14, 49–58; Shifman, M. I., and Selzer, M. E., 2000b. In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression. Journal of Neuroscience Methods 104, 19–25) that selective chemorepulsion might explain why some neurons are bad regenerators and others not. To explore the role of additional chemorepulsive axonal guidance molecules during regeneration, we examined the expression of the repulsive guidance molecule (RGM) and its receptor neogenin by in situ hybridization and quantitative PCR. RGM mRNA was expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells. Following spinal cord transection, RGM message was downregulated in neurons close (within 10 mm) to the transection at 2 and 4 weeks, although it was upregulated in reactive microglia at 2 weeks post-transection. Neogenin mRNA expression was unchanged in the brainstem after spinal cord transection, and among the identified reticulospinal neurons, was detected only in “bad regenerators”, neurons that are known to regenerate well never expressed neogenin. The downregulation of RGM expression in neurons near the transection may increase the probability that regenerating axons will regenerate through the site of injury and entered caudal spinal cord.     

Influences of Helicobacter pylori on cyclooxygenase-2 expression and prostaglandinE2 synthesis in rat gastric epithelial cells in vitro

BACKGROUND AND AIM: It is known that cyclooxygenase (COX)-2 is over expressed in gastrointestinal neoplasia and Helicobacter pylori (H. pylori) infection is causally linked to gastric cancer. The present study aimed to elucidate the effects of H. pylori on COX-2 expression and prostaglandinE(2) (PGE(2)) production in a gastric epithelial cell line derived from normal rat gastric mucosa (RGM1). METHOD: H. pylori water extracts were prepared from a supernatant of the H. pylori suspension in distilled water. RGM1 cells were cultured with H. pylori water extracts at the final concentration of 2.5, 5, 10 microg/mL for 24 h. For the time sequence study, RGM1 cells were cultured with 10 microg/mL H. pylori water extracts for 0, 6, 12, 24 and 48 h. COX-1 and COX-2 expression in the RGM1 cells was analyzed by western blotting. The levels of PGE(2) in the cultured media were measured by enzyme immunoassay. RESULTS: H. pylori did not affect COX-1 expression; whereas COX-2 expression increased by six-fold at 24 h after incubation of RGM1 cells with 10 microg/mL H. pylori water extracts. The increase in COX-2 expression was evident after 12 h of incubation; reached a peak at 24 h and declined at 48 h. H. pylori dose dependently increased COX-2 expression and PGE(2) synthesis in RGM1 cells. CONCLUSION: H. pylori induces COX-2 expression and increases PGE(2) synthesis in RGM1 cells in vitro. These results indicate that H. pylori-associated gastric carcinogenesis may depend on COX-2 expression.     

TGF-β superfamily co-receptors in cancer

Transforming growth factor-β (TGF-β) superfamily signaling via their cognate receptors is frequently modified by TGF-β superfamily co-receptors. Signaling through SMAD-mediated pathways may be enhanced or depressed depending on the specific co-receptor and cell context. This dynamic effect on signaling is further modified by the release of many of the co-receptors from the membrane to generate soluble forms that are often antagonistic to the membrane-bound receptors. The co-receptors discussed here include TβRIII (betaglycan), endoglin, BAMBI, CD109, SCUBE proteins, neuropilins, Cripto-1, MuSK, and RGMs. Dysregulation of these co-receptors can lead to altered TGF-β superfamily signaling that contributes to the pathophysiology of many cancers through regulation of growth, metastatic potential, and the tumor microenvironment. Here we describe the role of several TGF-β superfamily co-receptors on TGF-β superfamily signaling and the impact on cellular and physiological functions with a particular focus on cancer, including a discussion on recent pharmacological advances and potential clinical applications targeting these co-receptors.     

Pulmonary disease due to nontuberculous mycobacteria

Nontuberculous mycobacteria (NTM) are increasingly associated with pulmonary disease. This is a worldwide phenomenon and one that is not related just to better diagnostic techniques or HIV infection. The mode of transmission of NTM is not well defined, but environmental exposure may be the major factor. While most exposed and infected individuals never acquire NTM disease, some ostensibly immunocompetent persons will. Although our understanding of the pathogenesis of NTM disease is incomplete, we believe that both host and mycobacterial factors are involved. Among the former, interferon-gamma"trafficking" may well play a central role. When disease occurs, it is likely to present in one of three prototypical forms: a tuberculosis-like pattern often affecting older male smokers with COPD; nodular bronchiectasis classically occurring in middle-aged or older women who never smoked and present with cough; and hypersensitivity pneumonitis following environmental exposure. While Mycobacterium avium complex has been described with all three forms, many other NTM can produce one or another of them; variants of these prototypes also exist. Diagnosis of NTM disease relies on microbiology and chest CT scanning, and criteria to aid diagnosis are available. Treatment of disease depends on the species involved, extent and form of disease, and overall condition of the patient. Surgery for localized disease may be useful for those species expected to be refractory to medical therapy. Observation without treatment may be appropriate for some patients with slowly progressive disease that is expected to be particularly difficult to treat.     

Hydrogen peroxide-induced cellular injury is associated with increase in endogenous fluorescence from rat gastric mucosal epithelial cell culture: A new method for detecting oxidative cellular injury by fluorescence measurement

To develop a new method of detecting cellular injury caused by oxygen radicals, we studied endogenous fluorescence from the cultured cells of a rat gastric mucosal epithelial cell line. Measurement with an ultra-high sensitivity camera-image processor system under an inverted epifluorescence microscope showed that the fluorescence intensity of the cells increased time- and dose-dependently after the addition of hydrogen peroxide (H₂O₂), an oxygen radical precursor, to the medium. This increase was inhibited by the presence of catalase. Phase-contrast and fluorescence microscopy revealed that the fluorescence was emitted from granular substances in the cytoplasm of the injured cells. The spectral pattern of excitation and emission indicated that the fluorescent substances were flavins. In cell-free experiments, glutathione reductase which has flavin adenine dinucleotide (FAD) at the active site, increased in fluorescence after incubation with H₂O₂ in the presence of reduced glutathione and glutathione peroxidase. These findings indicate that FAD in the cytoplasm of cells injured by H₂O₂ increased in endogenous fluorescence according to the extent of injury, and suggest that fluorescence measurement may be a simple method in cellular toxicology to detect oxygen radical-induced injuries. (Received July 28, 1997; accepted Sept. 26, 1997)     

Impairment by activation of TRPA1 of gastric epithelial restitution in a wound model using RGM1 cell monolayer

We examined the influence of TRPA1 on the epithelial restitution using a rat gastric epithelial cell line RGM1 monolayer. RGM1 cells were inoculated in 24-well plates cultured for 24 hr, and then starved for 24 hr in a culture medium at 37 °C under 5 % CO₂ in air. After obtaining a confluent RGM1 cell monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. The repair process was monitored by quantitatively measuring the area of the epithelial wound (cell-free area). Immediately after the wound induction, cells at the edge of wound started to form lamellipodia, migrating toward the center of wound, and by so doing the cell-free area was decreased over time. The addition of icilin, the TRPA1 agonist, suppressed the recovery of the epithelial wound in a concentration-dependent manner. Likewise, another TRPA1 agonist, ally isothiocyanate, also significantly inhibited the wound repair. In addition, the recovery of the epithelial wound was potently inhibited when the ambient temperature was lowered to 17 °C, the threshold temperature where TRPA1 is known to be activated. By contrast, the wound healing was not affected by either menthol, the TRPM8 agonist, or capsaicin, the TRPV1 agonist. These results showed for the first time that the activation of TRPA1 inhibited the repair of the epithelial wound in the stomach, probably by the suppression of cell migration, and suggested the involvement of TRPA1 in the mechanism of gastric epithelial restitution. Received 8 August 2006; accepted 7 November 2006