Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line

Area of Science:

• Gastroenterology and Nutrition
• Molecular Biology
• Cell Biology

Background:

• The Caco-2 cell line serves as a validated model for studying intestinal epithelial transport.
• Epidermal Growth Factor (EGF) is a key regulator of cell growth and differentiation, with potential roles in intestinal transport.
• Peptide transporters, like hPepT1, are crucial for absorbing dietary peptides and certain drugs in the intestine.

Purpose of the Study:

• To investigate the effect of EGF on the transport of the dipeptide glycylsarcosine (Gly-Sar) in Caco-2 cells.
• To elucidate the molecular mechanisms underlying EGF-mediated changes in peptide transport.
• To assess the impact of EGF on the expression and function of the human peptide transporter 1 (hPepT1).

Main Methods:

• Utilized Caco-2 cell monolayers to measure apical-to-basolateral fluxes of radiolabeled glycylsarcosine ([14C]Gly-Sar).
• Performed kinetic analyses to determine maximal transport velocity (Vmax) and Michaelis-Menten constant (Km).
• Employed RT-PCR and Western blotting to quantify hPepT1 mRNA and protein levels, respectively.

Main Results:

• EGF treatment significantly reduced Gly-Sar transepithelial transport by up to 50.2%, primarily by decreasing Vmax.
• Apical uptake of Gly-Sar was inhibited by EGF, with an ED50 of 0.36 ng/ml, while Km remained unchanged.
• EGF decreased both hPepT1 mRNA and protein expression in Caco-2 cells, indicating reduced transporter availability.

Conclusions:

• EGF diminishes peptide transport in Caco-2 cells through a reduction in the number of apical hPepT1 transporters.
• This downregulation of hPepT1 by EGF has implications for understanding nutrient absorption and the efficacy of peptide-based drugs.
• The findings highlight EGF's role in regulating intestinal peptide transport at the molecular level.