Regulation of Breast Milk Induced P-Glycoprotein Expression in Intestinal Epithelial Cells

Introduction: P-glycoprotein (Pgp), a transmembrane efflux pump, is found at the apical surface of intestinal epithelial cells. Because adult Pgp-deficient mice develop colitis in a bacteria-dependent manner, and newborn Pgp-deficient mice display increased susceptibility to necrotizing enterocolitis (NEC), Pgp is believed to play a key role in protecting the intestinal epithelium against bacteria and their by-products. Pgp expression is induced by a variety of stresses and xenobiotics. the mechanisms by which these stimuli induce Pgp expression are not fully understood. Previous studies have shown that ß-catenin, PI3-kinase, c-jun and other signaling intermediates may be involved in the induction of Pgp. Recently we have demonstrated that expression of Pgp in the intestinal epithelium is upregulated by breast milk. We hypothesize that factor(s) in breast milk induce Pgp via their cognate receptors and intracellular signaling pathways. Our goal is to elucidate the signaling pathways by which breast milk upregulates Pgp expression. Methods: Human breast milk was obtained from healthy volunteers. the crude fraction containing the Pgp-inducing activity was obtained by precipitation with ammonium sulfate at 40% saturation. Casein was precipitated using high temperature centrifugation and dialysis at pH 4.6. Cultured enterocytes were grown to confluence at 37ºC in medium supplemented with 20% FBS. Cells were serum starved for 12 hours prior to treatments. Specific inhibitors of MEK, p38, PKA, PKC, JAK, B-catenin, and PI 3-Kinase were added 1 hour prior to treatment with the enriched fraction of breast milk. 18 hours post treatment, cells were lysed with RIPA buffer and Pgp expression was analyzed using western blotting with the C219 monoclonal antibody. Appropriate controls were used with each inhibitor. Results: Inhibition of MEK, JAK, PKC, and PI 3-Kinase appeared to have no effect on breast milk-induced expression of Pgp. Inhibition of ß-catenin and PKA partially suppressed expression of Pgp. in contrast, inhibition of p38, which has previously been shown to decrease Pgp expression, resulted in a marked potentiation of Pgp in our model. Conclusions: Breast milk-induced expression of Pgp in the intestinal epithelium, appears to be mediated by multiple pathways. ß-catenin and PKA pathways may act as positive regulators, whereas p38 may act as a negative regulator. Understanding the pathway of breast milk-induced expression of Pgp will further elucidate the mechanisms by which breast feeding protects against NEC.