Bovine Lactoferricin Induces Intestinal Epithelial Cell Activation through Phosphorylation of FAK and Paxillin and Prevents Rotavirus Infection

We investigated the effect of bovine lactoferricin (Lfcin-B), a peptide derived from bovine lactoferrin, on activation of intestinal epithelial cells in IEC-6 intestinal cell, and protection against in vivo rotavirus (RV) infection. Treatment with Lfcin-B significantly enhanced the growth of IEC-6 cells and increased their capacity for attachment and spreading in culture plates. Also, Lfcin-B synergistically augmented the binding of IEC-6 cells to laminin, a component of the extracellular matrix (ECM). In the analysis of the intracellular mechanism related to Lfcin-B-induced activation of IEC-6 cells, this peptide upregulated tyrosine-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin, which are intracellular proteins associated with cell adhesion, spreading, and signal transduction during cell activation. An experiment using synthetic peptides with various sequences of amino acids revealed that a sequence of 9 amino acids (FKCRRWQWR) corresponding to 17-25 of the N-terminus of Lfcin-B is responsible for the epithelial cell activation. In an in vivo experiment, treatment with Lfcin-B one day before RV infection effectively prevented RV-induced diarrhea and significantly reduced RV titers in the bowels of infected mice. These results suggest that Lfcin-B plays meaningful roles in the maintenance and repair of intestinal mucosal tissues, as well as in protecting against intestinal infection by RV. Collectively, Lfcin-B is a promising candidate with potential applications in drugs or functional foods beneficial for intestinal health and mucosal immunity.


Introduction
The gastrointestines can be described as epithelial-lined mucosal tissues that play an important role in nutrient absorption and host defense systems [1]. Interactions between enterocytes and the basal membrane are essential for cell adhesion, migration, differentiation, and maintenance of mucosal tissue integrity [2,3]. Cell adhesion triggers migration, proliferation, and differentiation [4,5], while the binding of cell surface molecules like integrin to the extracellular matrix (ECM) increases tyrosine phosphorylation of several focal adhesion proteins such as FAK (125 kDa) and paxillin (68 kDa) to bring about the assembly of actin filaments as well as cell proliferation [6,7].
Rotavirus (RV) is a major agent of viral gastroenteritis in various animal species. It is regarded as the leading cause of acute diarrhea in human infants and young children under the age of five and annually causes 215,000 deaths worldwide [8,9]. Thus, the mortality and morbidity of RV-associated diarrhea makes this virus an important pathogenic agent to be controlled. Current live vaccines can induce anti-diarrheal effect, but they are neither distributed around the world nor sufficiently effective in some developing countries [10]. Therefore, the development of alternative approaches to prevent RV-induced diarrhea is urgently needed. Since intestinal epithelial cells (IECs) are the primary responder to RV in the initial period of infection, the dysfunction of these cells has been considered as a major cause of diarrhea [11].
Breast milk has a lot of components that take charge of diverse physiological properties, such as nutritional source and host defense against pathogens, especially on mucosal surfaces of the gastrointestinal tract [12]. The iron-binding protein lactoferrin (LF), mainly found in body fluids like breast milk, is well known to have a diversity of biological functions, i.e., antimicrobial activity, regulation of immune responses [13,14] and transcriptional activation of cells [15]. Upon ingestion of LF, the protein is degraded by gastrointestinal enzymes in the stomach, like pepsin, and may create bioactive peptides like lactoferricin (Lfcin-B) [16,17]. Lfcin-B has been shown to be a key peptide responsible for the antibacterial activity of bovine LF [18,19]. Taken together, it is possible that Lfcin-B generated from pepsin lysate of LF arrives at the intestines, where it interacts with IECs to We investigated the effect of bovine lactoferricin (Lfcin-B), a peptide derived from bovine lactoferrin, on activation of intestinal epithelial cells in IEC-6 intestinal cell, and protection against in vivo rotavirus (RV) infection. Treatment with Lfcin-B significantly enhanced the growth of IEC-6 cells and increased their capacity for attachment and spreading in culture plates. Also, Lfcin-B synergistically augmented the binding of IEC-6 cells to laminin, a component of the extracellular matrix (ECM). In the analysis of the intracellular mechanism related to Lfcin-B-induced activation of IEC-6 cells, this peptide upregulated tyrosine-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin, which are intracellular proteins associated with cell adhesion, spreading, and signal transduction during cell activation. An experiment using synthetic peptides with various sequences of amino acids revealed that a sequence of 9 amino acids (FKCRRWQWR) corresponding to 17-25 of the N-terminus of Lfcin-B is responsible for the epithelial cell activation. In an in vivo experiment, treatment with Lfcin-B one day before RV infection effectively prevented RV-induced diarrhea and significantly reduced RV titers in the bowels of infected mice. These results suggest that Lfcin-B plays meaningful roles in the maintenance and repair of intestinal mucosal tissues, as well as in protecting against intestinal infection by RV. Collectively, Lfcin-B is a promising candidate with potential applications in drugs or functional foods beneficial for intestinal health and mucosal immunity.

RV Infection Experiment
RV (1.5 × 10 6 PFU/50 µl/mouse) was inoculated per os (p.o.) into the groups of seven 10-day-old Balb/c newborn mice. Mice were fasted for 4 h before RV infection [25]. Lfcin-B was administered p.o. 1 day before virus infection. Ginsenoside Rb2 was used as a positive control for protection against RV [20]. A clinical score of diarrhea induced by RV infection was determined by individual severity of diarrhea every 24 h after virus infection: point 2; serious, point 1; moderate, point 0; cured. The diarrhea score for each group was calculated as follows: (the number of mice under serious diarrhea) × 2 + (the number of mice under moderate diarrhea) × 1/ total number of mice [25]. The total diarrhea score was estimated as cumulative diarrhea scores obtained during the entire observation time.

RV Isolation from the Bowels
Virus titers in the bowels of RV-infected newborn mice were determined by plaque formation on MA-104 cells as described previously [20]. Briefly, the bowels removed from the mice were homogenized in 1 ml of EMEM. After centrifugation, the supernatants were massed up to 5 ml with EMEM, and stored at -80 o C until use. Each homogenate (0.5 ml/well) in 1,000-fold dilution was added onto the monolayer of MA-104 cells in 6-well culture plates and incubated at 37 o C for 1 h. After washing twice with EMEM, the cells were overlaid with 2.5 ml of EMEM containing 0.7% purified agar (Agarose; SeaKem ME, FMC BioProducts, USA) and 0.0001% trypsin, and incubated at 37 o C for 5 days. The cells were subsequently overlaid with EMEM (2 ml/well) containing 0.7% purified agar and 0.005% neutral red for 2 days. Thereafter, the plaques formed in RV-infected MA-104 cells were counted.

Statistical Analysis
The statistical significance was determined by Student's two-tailed t-test.

Effect of Lfcin-B on Cell Proliferation and Growth
IEC-6 cell, a non-transformed rat jejunum crypt cell line [26], has been widely employed in studies on the effect of various growth factors and cytokines on intestinal crypt cell growth and maturation [27,28]. To address the influence of Lfcin-B on the proliferation of intestinal cells, we examined its activity to enhance DNA synthesis of IEC-6 cells by [ 3 H]-TdR uptake assay. Treatment with Lfcin-B significantly increased DNA synthesis of IEC-6 cells in a dose-dependent manner, showing maximal activity from the dose of 50 µg/ml (Fig. 1A). However, BSA, a control protein, had no effect. When we examined the number of IEC-6 cells at incubation times of 36 and 48 h, the cells treated with Lfcin-B (50 or 100 µg/ml) showed a significant increase in cell number (Fig. 1B) from the incubation of 36 h. The results indicated that Lfcin-B actively augments the growth of IECs through acceleration of DNA synthesis.

Effect of Lfcin-B on Attachment and Spreading of IEC-6 Cells
Cell adhesion is a multistep process involving primary receptor-ligand interactions followed by secondary events that may lead to the formation of focal contacts and the expression of cell functions [29]. Generally, focal adhesion (attachment) is an important event during structural links between the cytoskeleton and extracellular matrix (ECM), and induction of signal transduction in adherent cells like IECs. To examine the influence of Lfcin- B on attachment of IECs, IEC-6 cells were treated with various doses of Lfcin-B for 30 min, and attached cells were estimated using cell staining with crystal violet. IEC-6 cells treated with Lfcin-B showed significantly higher attachment than that of negative control (BSA-treated) cells, and its enhancing effect was dose-dependent ( Fig. 2A). Maximal activity of Lfcin-B to enhance cell attachment was also observed from the dose of 50 µg/ml. Next, to elicit the effect of Lfcin-B on spreading of IECs, IEC-6 cells were incubated in the presence of Lfcin-B (50 µg/ml) or BSA as a negative control, and the spreading cells were examined at the incubation times of 30, 60 and 90 min. As shown in Fig. 2B, spontaneous spreading of IEC-6 cells increased with the lapse of incubation time. Meanwhile, the cells treated with Lfcin-B showed a significant increase of cell spreading at all incubation times. In addition, an inverted photomicrograph of cultured cells clearly showed that Lfcin-B promoted cell adhesion and spreading even at the early period (15 min) of incubation time (Fig. 3).
The epithelial basement membranes (BM) are ECM structures consisting of well-organized glycoproteins, particularly laminin (LN), fibronectin, and type IV collagen. These proteins regulate biological processes such as migration, proliferation, and differentiation of IECs [30]. Among ECM molecules, LN, one of the most abundant proteins present in the BM, can bind to IECs and modulate several biological functions of the cells, including cell growth, cell adhesion and migration, and gene expression [31]. When IEC-6 cells were incubated with Lfcin-B in LN-coated culture plates, treatment with Lfcin-B synergistically enhanced cell adhesion of the cells in a dosedependent manner (Fig. 4). This finding indicated that Lfcin-B can upregulate the biological and physiological  The intestinal epithelium is sometimes damaged by toxins or pathogenic microorganisms, and, in this case, mucosal restitution rapidly works to repair the damaged epithelium. This restoration work includes the peeling of damaged epithelial cells and the spreading and migration of viable cells to reconstruct epithelial continuity [32]. The intestinal mucosa is established by epithelial cells that become differentiated after migrating from the crypts to the villus. This means that activation of intestinal crypt cells may be directly connected to upregulation of maintenance and repair of intestinal mucosa [26]. The data from Fig. 1 to Fig. 4 consistently proved that Lfcin-B enhanced not only cell proliferation but also attachment and spreading of the intestinal crypt cell line, IEC-6. These results suggest that Lfcin-B can play roles in maintenance of the mucosal lining and repair of the damaged epithelium in the intestines.

Lfcin-B Induces Phosphorylation of FAK and Paxillin
Focal adhesion is a highly dynamic membrane-associated multi-protein structure that binds cells to the ECM and regulates various cellular and biological actions. FAK and paxillin are potential regulatory molecules involved in interactions between cellular proteins and extracellular molecules during focal adhesion and have been recognized as a critical link in the signal transduction pathway in adherent cells [6,33]. To examine whether the ability of Lfcin-B to activate IEC-6 cells is related to induction of focal adhesion in the cells, we examined phosphorylation of FAK and paxillin by Lfcin-B using an immunoprecipitation assay, in which cellular proteins were precipitated with anti-paxillin or anti-FAK antibody followed by immunoblotting with anti-phosphtyrosine antibody. Lfcin-B treatment prominently augmented tyrosine phosphorylation of FAK in IEC-6 cells, showing apparent enhancement from the early incubation period of 15 min and maximal effect at 60 min (Fig. 5A). Furthermore, IEC-6 cells treated with Lfcin-B indicated higher level of phosphorylated paxillin than that of untreated cells at 60 min (Fig. 5B). These results suggest that the activity of Lfcin-B to enhance attachment and spreading of intestinal IEC-6 epithelial cells is associated with phosphorylation of FAK and paxillin. Considering

Fig. 5. Effect of Lfcin-B on phosphorylation of FAK in IEC-6 cells. Phosphorylation of FAK in IEC-6 cells treated
with Lfcin-B (50 µg/ml) was measured by immunoprecipitation and western blot assays by the method described in Materials and Methods. PTK is an abbreviation for protein tyrosine kinase. Bar graphs represent the results of densitometry analysis. that, in general, the event of phosphorylation of intracellular proteins such as FAK and paxillin in adherent cells occurs during interactions with extracellular proteins like those of the ECM (Kolachala et al., 2007), it is significant that Lfcin-B acts as an inducer of cellular signals to result in cell proliferation and adhesion.

Minimal Sequence of Lfcin-B Responsible for Activation of IEC-6 Cells
Next, to determine the minimal structure of Lfcin-B responsible for its ability to induce IEC-6 cell activation, we synthesized many types of peptides containing the partial sequences of Lfcin-B, and investigated their ability to enhance cell proliferation. As seen in Fig. 6, the minimal sequence showing identical activity of the full length of Lfcin-B was 9 amino acids (FKCRRWQWR; P17-25). This result means that the nine amino-acid peptide from the N-terminus of Lfcin-B is a minimal peptide of Lfcin-B responsible for the activation of IEC-6 epithelial cells.
The present study demonstrated for the first time that Lfcin B, a peptide derived from the milk protein lactoferrin, is a novel peptide that induces activation of IEC-6 rat intestinal epithelial cells through phosphorylation of FAK and paxillin, and the minimal sequence responsible for its activity is FKCRRWQWR, which corresponds to nine amino acids from the N-terminus of Lfcin-B. These findings suggest that Lfcin-B is a biologically active milk protein-derived peptide that contributes to recovery of damaged intestinal epithelium and enhancement of host defense system in the gut.

Effect of Lfcin-B on RV Infection
Considering that IECs react first to RV at the onset of infection [11], the condition of the cells may be a very important factor influencing the occurrence of RV-induced acute diarrhea. Based on the results showing that Lfcin-B can activate IECs, we examined whether this peptide prevents the intestinal infection caused by RV. Oral administration of Lfcin-B 1 day prior to infection resulted in a highly protective effect against RV-induced acute diarrhea ( Table 1). The reason was unclear, but a lower dosage of Lfcin-B (50 µg) was more effective than a higher dosage (100 µg). RV is a virus that infects IECs at the initial stage of infection, proliferates, and then causes acute diarrhea [34]. Since it is important to prevent the growth of the virus in the early period of infection, we next investigated whether oral administration of Lfcin-B (50 µg) inhibits the intestinal proliferation of RV. In the newborn mouse infection model, RV showed the highest virus titer 15 h after infection, and then gradually decreased (Fig. 7). Oral administration of Lfcin-B (50 µg) significantly reduced RV titer in the intestines at least  15 h after infection. These results suggest that Lfcin-B has an inhibitory effect on RV infection and the antiviral effect of this peptide is due to suppression of the infection in epithelial cells at the initial stage of infection.
In this study, we demonstrated that Lfcin-B, a peptide derived from bovine lactoferrin, can activate IECs via upregulation of phosphorylation of FAK and paxillin, in in vitro experiments, and oral administration of this peptide also effectively prevented severe infection by RV in in vivo models. Taken together, it is strongly suggested that Lfcin-B is a potent stimulant to potentiate biological functions of the intestinal epithelial cells and nonspecific resistance against RV infection. RV infection is inhibited by immunological factors such as interferons produced by immune cells in the intestinal epithelial tissue; however, it remains unclear whether Lfcin-B-induced epithelial cell activation elicits the immunological response necessary to suppress RV virus infection. Currently, research is underway to elucidate the correlation between Lfcin-B-induced epithelial cell activation and its protective activity against RV infection.