EM-197---Intestinal trefoil factor activates PI3KAkt signaling pathway to protect gastric mucosal

Intestinal trefoil factor activates PI3K/Akt signaling pathway to protect gastric mucosal epithelium from damage

Zhaorui Sun1#, Hongmei Liu1#, Zhizhou Yang1, Danbing Shao1, Wei Zhang1, Yi Ren1, Baodi Sun1, Jinfeng Lin1, Min Xu1, Shinan Nie1*

1 Department of Emergency, Jinling Hospital, Medical School of Nanjing University, Nanjing, 210002, PR China.

Mailing address: Department of Emergency, Jinling Hospital, Medical School of Nanjing University, Zhongshan East Road 305, Nanjing, 210002, PR China. * Corresponding author:

Shinan Nie, shn_nie@http://wendang.chazidian.com, Department of Emergency, Jinling Hospital, Nanjing University School of Medicine, Zhongshan East Road 305, Nanjing, 210002, PR China.

# Co-first authors, they contributed equally.

Abstract

Intestinal trefoil factor (ITF, also named as trefoil factor 3, TFF3) is a member of the TFF-domain peptide family, which plays an essential role in regulating cell survival, cell migration and maintains mucosal epithelial integrity in gastrointestinal tract. However, the underlying mechanisms and associated molecules remain unclear. The aim of this study was to explore the protective effects of ITF on gastric mucosal epithelium injury and its possible molecular mechanism. In the present study, we show that ITF could promote proliferation and migration of GES-1 cells via a mechanism that involves the PI3K/Akt signaling pathway. Western blot results indicated that ITF induced a dose- and time-dependent increase in Akt signaling pathway. ITF also plays an essential role in restitution of GES-1 cells damage induced by LPS (Lipopolysaccharide). LPS induced GES-1 cells apoptosis，decreased cell viability significantly (P <0.01) and led epithelial tight junction damage which is attenuated via ITF treated. The protective effect of ITF on the integrity of GES-1 was abrogated by inhibition of the PI3K/Akt pathway. Taken together, our results demonstrate that ITF promotes proliferation and migration of gastric mucosal

epithelial cells and preserves gastric mucosal epithelial integrity after damage is mediated by activation of PI3K/Akt signaling pathway. This study suggested that PI3K/Akt pathway could act as a key intracellular pathway in the gastric mucosal epithelium that may serve as a therapeutic target to preserve epithelial integrity during injury.

Key words: Intestinal trefoil factor; mucosal epithelium; integrity; PI3K/Akt signaling pathway

Introduction

Various traumas on body can cause gastrointestinal tract erosion and mucosal epithelium damage which lead gastrointestinal tract bleeding and ulcer perforation finally to aggravate origin disease. The gastrointestinal mucosal epithelium is a fundamental barrier that provides protection against the outside environment. It is important to protect the mucosal epithelium from damage. The cytoprotective functions in protecting gastrointestinal tract against ongoing damage may be accomplished in both the early phase of epithelial repair known as restitution and in the subsequent, protracted phase of epithelial renewal [1-3]. Restitution, the ability of epithelial cells to spread and migrate across the basement membrane to cover shallow defects, is the key initial step in repair of mucosal injury and can achieve restoration of mucosal continuity over broad areas of damage within hours [4, 5]. It is well established that the integrity of the gastrointestinal mucosa and other epithelium are maintained by a number of secreted factors, but many studies suggest that one important mechanism involves the secretion of the members of a protease-resistant protein family known as the trefoil factor family (TFF) [6].TFF, a recently recognized family of protease-resistant small peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. This family, comprising the intestinal trefoil factor (ITF) and the gastric peptides SP and pS2, play a critical role in epithelial restitution and proliferation within the mammalian gastrointestinal tract [7]. The members of this family share an array of structural features including, most notably, a motif of six cysteine residues termed a trefoil or a P domain, which is distinct from those found in other peptide families. They are rapidly up-regulated at

the margins of mucosal injury, and they are believed to promote epithelial cell proliferation and migration [8, 9]. ITF is a predominant factor of TFF and has been demonstrated in in vitro and in vivo studies to play an important role in mucosal homeostasis of the gastrointestinal mucosa [10, 11]. Though ITF could maintain gastric mucosal integrity, the underlying mechanisms controlling this process remain unclear yet. It is necessary to explore the mechanisms of ITF that regulate the proliferation, migration and maintenance integrity of gastric mucosal epithelial cells. It is well known that phosphatidylinositol 3´-kinase (PI3K) is a vital regulatory protein responsible for maintaining cell viability and cell survival. PI3K phosphorylates phosphoinositides at their 3´-position in turn, activates downstream effector molecules. Akt/protein kinase B is a primary downstream target of PI3K intermediates and major component in cell survival. Akt is activated by translocation to plasma membrane when the PI3K-generated 3-phosphoinositides bind to its pleckstrin homology domain. PI3K/Akt signaling pathway plays an essential role in regulating cell proliferation, differentiation, apoptosis and migration [12, 13]. In addition to its metabolic actions, PI3K/Akt signaling pathway has been shown to preserve epithelial integrity during inflammation [14]. Numerous studies have indicated that growth factors participate in many important physiological and pathologic processes in gastrointestinal diseases via regulating downstream signaling pathways which could mediate cell survival, cell apoptosis, cell migration and immune response. Evidence has shown that ITF (TFF3) as a growth factor could act on the epidermal growth factor receptor (EGFR), which then activates several downstream signaling pathways, including MAPK and PI3K/Akt signaling pathway

[15]. The previous study has also demonstrated that ITF exerts antidepressant-like effects that might be mediated by the PI3K/Akt signaling pathway in the basolateral amygdala. These studies suggested that PI3K/Akt signaling may participate in the regulation of ITF on the cell physiological and pathologic processes. Based on these observations, we hypothesized that ITF could activate the PI3K/Akt signaling to regulate the proliferation, migration and maintain epithelial integrity of GES-1 in vitro.

The present study examined the effects of ITF on the proliferation, migration and epithelial integrity of GES-1 cells in vitro. To investigate the underlying mechanisms, we evaluated the role of the PI3K/Akt signaling pathway in these physiological processes regulated by TFF3. Thus, we intend to observe the protective effect of ITF on the gastric mucosa epithelium after damage to lay the foundations of clinical application of ITF as a new type of gastric mucosal protective agent.

Materials and methods

Cell culture

GES-1 cells were obtained from the A.T.C.C. (Rockville, MD, USA). Cells were cultured in high DMEM with 10% FBS, 1% L-glutamine, and a 1% solution of penicillin and streptomycin seeded at a density of 2×106 cells/ml onto uncoated flasks, and cultured in a humidified incubator at 37 °C in 5% CO2.

Cell viability assay

Cells were placed on a 96-well culture plate at 2×104 cells/ well in 100 μl culture medium. After incubation for 12 h, the wells were treated with ITF (100ng/ml), LY294002 (15μM) or LPS (10μg/ml) separately. The culture medium was removed and 100 μl free-serum medium was added with 10 μl CCK-8 solution to each well of culture plate. After incubated for 4 h, absorbance was measured optical density (OD) at 450 nm with a multi- detection micro plate reader.

Cell migration assay

Cells were suspended in serum-free culture medium at a concentration of 4×105 cells/ml. Simultaneously, 0.5 ml of culture medium with 10% FBS containing ITF (100ng/ml) or LY294002 (15μM) was added to the lower compartment. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 90% ethanol for 30 min at room temperature, washed three times with distilled water, and stained with 0.1% Crystal Violet in 0.1 M borate and 2% ethanol for 30 min at room temperature. Cells remaining on the upper surface of the filter membrane were gently scraped off with a cotton swab. Images of penetrated cells were captured by a photomicroscope. Cell migration ability was quantified in a blinded manner by counting the number of the penetrated cells on the lower surface of the membrane

with five fields (100× magnification) per chamber. Experiments were performed three times in duplicates.

Western blotting analysis

GES-1 cells were first treated with increasing doses of ITF (0–500 ng/ml) for 30 min. Next, the optimum dose (100 ng/ml) was used to determine the time-dependent response of Akt activation. Cell proteins were obtained from cultured or treated GES-1 cells in each group. Western blot analysis of cellular lysates was performed as previously described [22].

FDA and PI staining for morphologic evaluation

The integrity of cell membrane was detected using FDA/PI staining. Cells were placed on a 6-well culture plate at 1×106 cells/ml in 2 ml culture medium. After 12 h, cells were treated with LPS (10μg/ml), ITF (100ng/ml) and LY294002 (15μM) for 24 h, and then plated at a density of 2×105 cells/well onto 96-well plates, stained with 5 μg/ml PI and 4 μg/ml FDA, and observed under a fluorescent microscope. Flow cytometric assay

Cell apoptosis was measured by Annexin V-FITC and propidium iodide (PI) staining through flow cytometry. The cells were transplanted to 6-well culture plates at a density of 2×106 cells/ml, after 12 h, the cells were treated with LPS (10μg/ml), ITF (100ng/ml) and LY294002 (15μM), then cells were incubated for 48 h. After the cells were washed twice with cold PBS, they were re-suspended in 500 μl binding buffer at a concentration of 1×106 cells/ml. Each cell sample was then stained with 5 μL Annexin V-FITC and 5μL PI according to the manufacturer’s instructions. Treated cells were then incubated in the dark at 37 ?C for 15 min. Samples were acquired on a FACScan flow cytometer and analyzed with software.

Immunofluorescent staining

For studying the protective effect of ITF on maintaining integrity of GES-1 cells and investigating the regulation of Akt signaling pathway, 10μg/ml LPS was added into cultured GES-1 cells. After treated 4 h, 100ng/ml ITF were added, then treated GES-1 cells were cultured in this condition for 48 h. Immunofluorescence analysis of GES-1 cells was performed as described previously [16].