The proliferative effects of Pyropia yezoensis peptide on IEC-6 cells are mediated through the epidermal growth factor receptor signaling pathway

For a number of years, seaweed has been used as a functional food in Asian countries, particularly in Korea, Japan and China. Pyropia yezoensis is a marine red alga that has potentially beneficial biological activities. In this study, we examined the mechanisms through which a Pyropia yezoensis peptide [PYP1 (1–20)] induces the proliferation of IEC-6 cells, a rat intestinal epithelial cell line, and the involvement of the epidermal growth factor receptor (EGFR) signaling pathway. First, cell viability assay revealed that PYP1 (1–20) induced cell proliferation in a concentration-dependent manner. Subsequently, we examined the mechanisms responsible for this induction of proliferation induced by PYP1 (1–20). EGFR is widely expressed in mammalian epithelial tissues, and the binding of this ligand affects a variety of cell physiological parameters, such as cell growth and proliferation. PYP1 (1–20) increased the expression of EGFR, Shc, growth factor receptor-bound protein 2 (Grb2) and son of sevenless (SOS). EGFR also induced the activation of the Ras signaling pathway through Raf, MEK and extracellular signal-regulated kinase (ERK) phosphorylation. In addition, cell cycle analysis revealed the expression of cell cycle-related proteins. The results demonstrated an increased number of cells in the G1 phase and an enhanced cell proliferation. In addition, the upregulation of cyclin D, cyclin E, Cdk2, Cdk4 and Cdk6 was observed accompanied by a decreased in p21 and p27 expression. These findings suggest that PYP1 (1–20) stimulates the proliferation of rat IEC-6 cells by activating the EGFR signaling pathway. Therefore, PYP1 (1–20) may be a potential source for the development of bio-functional foods which promotes the proliferation of intestinal epithelial cells.

Cell proliferation is induced through intracellular signal transduction mediated by receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR) (8). RTKs are cell membrane receptors for growth factors and other extracellular ligands. RTKs mediate cellular tyrosine phosphorylation and regulate intracellular signaling pathways, such as those involved in cell migration, differentiation and proliferation (9)(10)(11). RTKs of the EGFR family mediate essential cellular functions, including the regulation of cellular proliferation, growth, survival, migration, differentiation and development in normal and pathological states (12,13). The binding of EGF to EGFR initiates a number of molecular events. The EGF-EGFR molecular interaction activates growth-promoting signals primarily through the activation of Ras, leading to activation of the Ras/Raf/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways, as well as many others (14,15). EGFR is stimulated by the guanine nucle-otide-exchange factor, son of sevenless (SOS)-growth factor receptor bound protein 2 (Grb2) complex. This activation of EGFR leads to the activation of Ras. The Src homology 2 (SH2) domain of Grb2 binds to autophosphorylation sites of EGFR, including Y1068, and several other receptors. The SH3 domains of Grb2 bind to the proline-rich C-terminal domain of SOS, a guanine nucleotide exchange factor (GEF) for Ras. In this way, the SOS-Grb2 interaction plays a critical role in regulating the activation of Ras (16,17). The small guanosine triphosphatase protein Ras/MAPK signaling pathway is essential for the regulation of a variety of biological processes, such as cell growth, cell cycle, cell proliferation and cell senescence, all of which are important for normal development (18). It consists of a core module of three kinases comprising Raf, MEK and extracellular signal-regulated kinase (ERK) that transmit signals downstream of the small GTPase, Ras. GTP-loaded Ras triggers the sequential activation of Raf, MEK and ERK to promote cell survival and various cellular functions (19,20). Therefore, EGFR and related proteins are attractive targets affecting cell proliferation.
Western blot analysis. To prepare whole cell extracts, the IEC-6 cells in 100-mm dishes were cultured to 50-60% confluence and then incubated in SFM for 4 h. Fresh SFM containing PYP1 (1-20) (125, 250, 500 and 1,000 ng/ml) was added to the cells and incubated another 24 h, after which the cells were washed with phosphate-buffered saline (PBS) and suspended in extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% Na-deoxycholate, 1% NP-40 and 1 mM EGTA) containing protease inhibitors (1 mM Na 3 VO 4 , 1 µg/ ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM NaF and 1 mM PMSF) on ice. The extracts were centrifuged at 14,000 rpm for 10 min, and the supernatant was used in western blot analysis. Boiling sample buffer (30 µg) was added to the total cell lysate, and the samples were boiled for 10 min at 100˚C. Proteins were separated by 7.5-12.

Discussion
Many types of seaweed have received a great deal of attention from researchers in recent years for their high levels of nutrients, such as proteins, minerals, vitamins and polysaccharides.
PYP1 (1-20)-induced cell proliferation was examined by cell cycle analysis (Fig. 4). Treatment with PYP1 (1-20) markedly increased the proportion of cells in the G0/G1 phase from 47.6 to 64.4%, suggesting that PYP1 (1-20) promotes cell cycle progression (Fig. 4). The PYP1 (1-20)-induced cell cycle progression resulted in cell proliferation and was related to the expression of cell cycle-related proteins, such as cyclin and Cdk. The expression levels of cyclin D1, cyclin E, Cdk2, Cdk4, Cdk6, pRb, p21 and p27 were measured by western blot analysis. The expression levels of cyclin D1, cyclin E, Cdk2, Cdk4, Cdk6 and pRb were increased in a dose-dependent manner, whereas the expression levels of p21 and p27 decreased in a dose-dependent manner (Fig. 5). Thus, the PYP1 (1-20)-induced cell cycle progression resulted in IEC-6 cell proliferation.
In the present study, we demonstrate that PYP1 (1-20) mediates cell proliferation through an EGFR signaling pathway in IEC-6 cells. These findings suggest the significant role of EGFR in intestinal epithelial cell proliferation, as well as the potential role of PYP1 (1-20) as a bio-functional food with a proliferative effect on rat intestinal epithelial cells.