Epidermal growth factor receptor-dependent PI3K-activation promotes restitution of wounded human gastric epithelial monolayers.

Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.

Specific signaling cascades involved in cell spreading during healing of micro-wounded gastric epithelial monolayers.

Mechanisms that specifically modulate cell spreading and/or cell migration following epithelial wounding are poorly understood. Using micro-wounded human gastric epithelial monolayers, we show herein that EGF and TGFalpha maximally increase spreading of epithelial sheets under a cell proliferation-independent mechanism. Treatment of confluent HGE-17 cells with the phosphatidylinositol 3-kinase inhibitor, LY294002, and the epidermal growth factor receptor inhibitor, PD153035, strongly reduced basal and TGFalpha-stimulated cell spreading. While pharmacological inhibition of pp60src-kinase activity also attenuated basal epithelial spreading, addition of the mTOR/p70S6K inhibitor rapamycin or a specific siRNA targeting ILK sequence did not affect the kinetic rates of wound closure. Epithelial wound healing was initiated by actin purse-string contraction followed by lamellae formation. Conversely, disruption of actin and tubulin stability with cytochalasin D and nocodazole, respectively, inhibited epithelial sheet spreading. Finally, antibodies directed against the alpha3 integrin subunit, but not against the alpha6 or alpha2 subunits, attenuated epithelial sheet spreading as well as lamellae formation. In conclusion, the current investigation establishes that EGF/TGFalpha and the alpha3beta1 integrin, pp60c-src, EGFR and PI3K pathways are mainly associated with the cell spreading of the restitution process during healing of human gastric epithelial wounds.

Isolation and functional studies of human fetal gastric epithelium in primary culture.

Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and devoid of glandular zymogenic chief cells. We successfully applied a new nonenzymatic procedure using Matrisperse Cell Recovery Solution to dissociate the entire epithelium from human fetal stomach. Cultures were generated by seeding multicellular aggregates prepared by mechanical fragmentation. We further demonstrate that this simple and convenient technique allows for the maintenance of heterogenous gastric epithelial primary cultures on plastic without a biological matrix as well as the persistence of viable chief cells able to synthesize and secrete gastric digestive enzymes, i.e., pepsinogen and gastric lipase. In wounding experiments, epithelial restitution occurred in serum-reduced conditions and was modulated by exogenous agents. This culture system is thus representative of the foveolus-gland axis and offers new perspectives to establish the influence of individual growth factors and extracellular matrix components as well as their combinatory effects on gastric epithelium homeostasis.

Establishment of human gastric epithelial (HGE) cell lines exhibiting barrier function, progenitor, and prezymogenic characteristics.

The unavailability of human cell lines representative of the gastric glandular epithelium while able to form a functional barrier restricts the application of a cell culture approach to the field of gastric epithelial physiology. In the current study, we have characterized new non-transfected clones isolated from gastric carcinoma cell lines known to express functional markers of the human gastric mucosa (J Cell Biochem 2001;81:241). Twenty-one clones exhibiting epithelial-type junctions (renamed HGE cell lines) were isolated from NCI-N87 (ATCC CRL 5822), whereas only squamous cell lines could be generated from other native strains. Of these 21 clones, HGE-17 and HGE-20 formed dense coherent monolayers and displayed true epithelial phenotype. E-cadherin and ZO-1 proteins were consistently localized at the periphery of all cells which also generated transepithelial electrical resistance. Moreover, growth factors known to be trophic for the gastric mucosa were able to stimulate mitogenesis at subconfluence. HGE-17 exhibited a poorly differentiated precursor-like status and responded strongly to EGF/TGFalpha treatment in restitution assays. HGE-20 cells, on the other hand, exhibited a higher degree of differentiation at the ultrastructural level as well as higher gastric lipase and pepsinogen levels. These latter zymogens were compartmentalized into granules which also contained mucin-6 (MUC6, prezymogenic-like status). Exogenous hormones, i.e., 1 mug/ml hydrocortisone and 5 microM retinoic acid, significantly increased enzyme levels in HGE-20. In conclusion, HGE-17 and HGE-20 represent the first human gastric cell lines with true epithelial characteristics, opening a venue to important applications for the study of re-epithelization, permeability, and regulation of digestive functions in the context of gastric physiology and pathology.

Differential growth factor induction and modulation of human gastric epithelial regeneration.

While several autocrine/paracrine growth factors (GFs) can all stimulate epithelial regeneration in experimentally wounded primary gastric cultures, clinical relevance for their non-redundant cooperative actions in human gastric ulcer healing is suggested by the sequential pattern of GF gene induction in vivo. Using new HGE cell lines able to form a coherent monolayer with tight junctions as well as using primary human gastric epithelial cultures, we show that EGF, TGFalpha, HGF and IGFs accelerate epithelial restitution upon wounding, independently of the TGFbeta pathway (as opposed to intestinal cells). However, they differently modulate cell behavior: TGFalpha exerts strong effects (even more than EGF) on cytoplasmic spreading and non-oriented protruding activity of bordering cells whereas HGF preferentially coordinates single lamella formation, cell elongation and migration into the wound. IGF-I and IGF-II rather induce the alignment of bordering cells and maintain a compact monolayer front. The number of mitotic cells maximally increases with EGF, followed by TGFalpha and IGF-I,-II. The current study demonstrates that GFs differentially regulate the regeneration of human gastric epithelial cells through specific modulation of cell shape adaptation, migration and proliferation, further stressing that a coordination of GF activities would be necessary for the normal progression of post-wounding epithelial repair.

Laminins and TGF-beta maintain cell polarity and functionality of human gastric glandular epithelium.

The human gastric glandular epithelium produces a gastric lipase enzyme (HGL) that plays an important role in digestion of dietary triglycerides. To assess the involvement of extracellular matrix components and transforming growth factor-beta1 (TGF-beta1) in the regulation of this enzymic function, normal gastric epithelial cells were cultured on collagen type I, Matrigel, and laminins (LN)-1 and -2 with or without TGF-beta1. Epithelial morphology and HGL expression were evaluated using microscopy techniques, enzymic assays, Western blot, Northern hybridization, and RT-PCR. A correlation was observed between the cell polarity status and the level of HGL expression. TGF-beta1 alone or individual matrix components stimulated cell spreading and caused a downfall of HGL activity and mRNA. By contrast, Matrigel preserved the morphological features of differentiated epithelial cells and maintained HGL expression. The combination of LNs with TGF-beta1 (two constituents of Matrigel) exerted similar beneficial effects on epithelial cell polarity and evoked a 10-fold increase of HGL levels that was blunted by a neutralizing antibody against the alpha(2)-integrin subunit and by mitogen-activated protein kinase (MAPK) inhibitors PD-98059 (p42/p44) or SB-203580 (p38). This investigation demonstrates for the first time that a powerful synergism between a growth factor and basement membrane LNs positively influences cell polarity and functionality of the human gastric glandular epithelium through an activation of the alpha(2)beta(1)-integrin and effectors of two MAPK pathways.

Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.

The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.

Stomach regional specification requires Hoxa5-driven mesenchymal-epithelial signaling.

The genetic control of gut regionalization relies on a hierarchy of molecular events in which the Hox gene family of transcription factors is suspected to be key participant. We have examined the role of Hox genes in gut patterning using the Hoxa5(-/-) mice as a model. Hoxa5 is expressed in a dynamic fashion in the mesenchymal component of the developing gut. Its loss of function results in gastric enzymatic anomalies in Hoxa5(-/-) surviving mutants that are due to perturbed cell specification during stomach development. Histological, biochemical and molecular characterization of the mutant stomach phenotype may be compatible with a homeotic transformation of the gastric mucosa. As the loss of mesenchymal Hoxa5 function leads to gastric epithelial defects, Hoxa5 should exert its action by controlling molecules involved in mesenchymal-epithelial signaling. Indeed, in the absence of Hoxa5 function, the expression of genes encoding for signaling molecules such as sonic hedgehog, Indian hedgehog, transforming growth factor beta family members and fibroblast growth factor 10, is altered. These findings provide insight into the molecular controls of patterning events of the stomach, supporting the notion that Hoxa5 acts in regionalization and specification of the stomach by setting up the proper domains of expression of signaling molecules.