Epidermal growth factor and/or growth hormone induce differential, side-specific signal transduction protein phosphorylation in enterocytes.

BACKGROUND: Epidermal growth factor (EGF) plus growth hormone (GH) enhances luminal glutamine transport into rabbit and human intestinal cells. Our objective was to screen for activation status of signal proteins in C2(BBe)1 cells (enterocyte-like cell line) in response to side-specific EGF or GH treatment and to investigate the dependence of EGF receptor (EGFR) phosphorylation status on its tyrosine kinase. METHODS: C2(BBe)1 cells on Transwells were treated for 15 minutes on either the basolateral or apical-side with EGF or GH. Lysates underwent Kinetworks phospho site-screen-2.1 analysis (duplicate experiments). In addition, lysates from cells treated as above with or without tyrphostin AG1478 (a specific EGFR tyrosine kinase inhibitor) underwent Western blot analysis for total EGFR and EGFR phosphorylated on tyrosine 1173, 1086 or 1068 (4-7 experiments). RESULTS: Kinetworks phospho-screening demonstrated a broad range of interactions dependent on both side of exposure and protein studied. From this screen, it appears that ErbB2, Met, and insulin receptor (R)/insulin-like growth factor 1 R are not involved in the growth factors signals. For EGFR phosphorylation, basolateral, but not apical, EGF was a strong activator. Synergism was seen, but only with apical EGF plus basolateral GH. All EGFR phosphorylations were EGFR tyrosine kinase dependent. In contradistinction, apical EGF phosphorylated FAK and MAPKs. CONCLUSIONS: Kinetworks phosphoprotein screens can suggest pathways involved in side-specific and synergistic interaction between EGF and GH. For EGFR, synergism by EGF + GH was noticed only with Ap EGF plus Bl GH and was EGFR tyrosine kinase dependent. Adaptive intestinal responses due to enterally administrated EGF might be accelerated by the availability of parenteral GH.

Growth factor regulation of enterocyte nutrient transport during intestinal adaptation.

BACKGROUND: Intestinal adaptation occurs in response to injury or alteration in nutrient availability. It is both morphologic and physiologic in nature and can be mediated by growth factors and nutrients. Pathologic conditions such as short-bowel syndrome and inflammatory bowel disease lead to derangements in nutrient absorption that may exceed the body's regenerative and adaptive capacity. Failure to fully adapt often results in long-term dependence on parenteral nutrition, leading to decreased quality of life and excessive medical expenses. The therapeutic use of appropriate growth factors may increase the adaptive capabilities of the gut. DATA SOURCE: Medline and current literature review. CONCLUSIONS: The major known nutrient transporters present in the gut and the mechanisms by which growth factors alter transport activity during intestinal adaptation are summarized. Growth factors have the potential to improve nutrient absorption in some bowel diseases.

Pathogenesis of Barrett's esophagus: bile acids inhibit the Notch signaling pathway with induction of CDX2 gene expression in human esophageal cells.

BACKGROUND: Barrett's esophagus (BE) is the predominant risk factor for the development of esophageal adenocarcinoma. BE is characterized by intestinal metaplasia with goblet cells. Reflux of bile acids is known to induce intestinal metaplasia, but the mechanisms are unclear. Inhibition of Notch signaling accompanied by increased Hath1 and induction of caudal homeobox 2 (CDX2) may be involved in development of intestinal goblet cells. METHODS: Esophageal adenocarcinoma cell lines OE19 and OE33 were exposed for up to 8 hours to DCA (100-300 microM), and for up to 24 hours with and without the gamma-secretase inhibitor, DAPT (20 microM). Notch signaling components and CDX2 levels were measured by real-time PCR (for mRNA) and by Western blot analysis (for proteins). RESULTS: DCA induced a time and concentration dependent decrease in Notch pathway components mRNAs in OE33 and in the proteins in both cell lines. CDX2 mRNA and Hath1 protein were increased in OE19 by 3-fold. Inhibition of Notch pathway by DAPT decreased downstream Notch signaling mRNAs and proteins in both cell lines and increased Hath1 and CDX2 proteins only in OE19. CONCLUSION: Bile acid inhibition of Notch signaling in esophageal cells is correlated with an increase in Hath1 and CDX2 and may be one of the key processes contributing to the formation of BE.

Bile acid alone, or in combination with acid, induces CDX2 expression through activation of the epidermal growth factor receptor (EGFR).

OBJECTIVES: Bile acids and acid are implicated in the development of Barrett's esophagus. Evidence suggests that Barrett's esophagus intestinal metaplasia may occur via induction of caudal homeobox gene 2 (CDX2). We hypothesized that induction of CDX2 by bile acids may be due to ligand-dependent transactivation of epidermal growth factor receptor (EGFR). METHODS: Human mucosal epithelial cells (SEG-1) were treated for 0 to 24 h with up to 300 microM deoxycholic acid (DCA) at pH 7 or 5 with or without (w/wo) antibodies against EGFR ligand-binding site (Mab528, 3-5 mug/ml). Treatment with 100 ng/ml EGF served as control. CDX2 mRNA expression was determined by real-time polymerase chain reaction. EGFR activation was analyzed by Westerns of phosphorylated EGFR tyrosines. RESULTS: Acid (pH 5) increased the induction of CDX2 mRNA expression caused by DCA. CDX2 mRNA induction was markedly reduced by EGFR blockade with Mab528. Each treatment (pH 5, DCA or pH 5 plus DCA) activated the EGFR on all tyrosines tested but in different time courses. Phosphorylation by DCA was inhibited by Mab528. Activation of EGFR by DCA at pH 5 resulted in EGFR degradation, while that by DCA alone did not. CONCLUSION: Thus, CDX2 induction by DCA w/wo acid occurs through ligand-dependent transactivation of the EGFR. The variations in EGFR degradation pattern with DCA or DCA at pH 5 indicate differential transactivation pathways. The molecular pathogenesis of Barrett's esophagus may occur via bile-stimulated cell signaling through the EGFR.

In human entrocytes, GLN transport and ASCT2 surface expression induced by short-term EGF are MAPK, PI3K, and Rho-dependent.

Glutamine, a key nutrient for the enterocyte, is transported among other proteins by ASCT2. Epidermal growth factor (EGF) augments intestinal adaptation. We hypothesized that short-term treatment of human enterocytes with EGF enhances glutamine transport by increasing membranal ASCT2. To elucidate EGF-induced mechanisms, monolayers of C2(BBe)1 w/wo siRho transfection were treated w/wo EGF and w/wo tyrphostin AG1478 (AG1478), wortmanin, or PD98059. Total and system-specific (3)H-glutamine transports were determined w/wo 5 mmol/l amino acid inhibitors. Total and membranal ASCT2 proteins were measured by Westerns. EGF doubled glutamine transport by increasing B(0)/ASCT2 and B(0,+) activities. Despite the doubling of membranal ASCT2 protein with EGF treatment, total ASCT2 did not change. The increases in B(0)/ASCT2 activity and ASCT2 protein were eliminated by AG1478, PD98059, wortmanin, and siRho, while transport by B(0,+) was inhibited only by PD98059 and siRho. Thus, differential pathways are involved in EGF-induced increase in B(0)/ASCT2 glutamine transport and membranal ASCT2 compared to those involved in B(0,+) activity.

Growth hormone and epidermal growth factor upregulate specific sodium-dependent glutamine uptake systems in human intestinal C2BBe1 cells.

Glutamine (Gln) is one of the major oxidative fuels of the enterocyte and enters from the lumen via Na(+)-dependent transport mechanisms. When given parenterally, growth hormone (GH) + epidermal growth factor (EGF) increase apical Gln uptake after massive enterectomy in rabbits. Although both receptors are basolateral, GH and EGF are present in luminal contents. We hypothesized that short-term luminal growth factor exposure to enterocytes increases apical Gln uptake by selective upregulation of systems A, B(0,+), or ASC+B(0). A monolayer of C2(BBe)1 cells was exposed for 10 or 60 min to GH (500 microg/L), EGF (100 microg/L), both, or neither. Initial uptake of [(3)H]Gln (50 micromol/L) was measured in the presence of Na(+) or choline. The contributions of systems A, B(0,+), and ASC+B(0) were determined by competitive inhibition with arginine and/or alpha-(methylamino)butyric acid. Gln uptake was linear for up to 8 min. Na(+)-independent transport was negligible. Under control conditions the relative contributions of systems A, B(0,+), and ASC+B(0) were 0, 19 +/- 6, and 80 +/- 4%, respectively. GH alone had no effect on Gln transport. After 10 min of EGF exposure, Na(+)-dependent Gln uptake increased by 50% (P < 0.001) with no change in individual transport systems. Combined EGF and GH for 60 min increased Gln transport by system B(0,+) nearly 250% (P < 0.001) and system A from undetectable levels to 16% of total transport (P < 0.01). Thus, short-term luminal exposure to EGF+GH increases Na(+)-dependent Gln transport mainly by upregulating system B(0+).

Methods used to study intestinal nutrient transport: past and present.

BACKGROUND: Vitally important to the future of surgical care is the study of nutrition and nutrient uptake. Advances in this field of research have become increasingly dependent upon the disciplines of immunology, histology, and molecular biology. The fusion of these sciences has deepened our insight into the relationship between molecular structure and physiologic function. The ability to apply new technologies to this endeavor will enable the surgeon-investigator to further widen our understanding of nutrient transport. MATERIALS AND METHODS: Medline and current literature review. RESULTS AND CONCLUSIONS: We summarize many of the methods used to measure the uptake of nutrients by the intestinal epithelium, providing a historical perspective.

Comparison of tracheal aspirate and bronchoalveolar lavage specimens from premature infants.

Lung fluid obtained by tracheal aspiration (TA) or bronchoalveolar lavage (BAL) has been used to study bronchopulmonary dysplasia (BPD). These two sample collection methods have seldom been compared. Paired BAL and TA specimens were collected 1, 3, 7 and 28 days after birth in 40 infants <34 weeks' gestation during a randomized, controlled trial of dexamethasone for BPD prophylaxis. Interleukin 8 (IL-8) and cell counts were measured. Compared to subjects without BPD, those who developed BPD or died by 28 days had elevated IL-8 in epithelial lining fluid on day 1 in both BAL specimens (20 ng/ml vs. 2 ng/ml) and TA specimens (101 ng/ml vs. 18 ng/ml). IL-8 levels (r = 0.55) and neutrophil proportions (r = 0.51) were moderately correlated between BAL and TA samples. TA specimens may be suitable substitutes for BAL samples in some studies of newborn lung fluid.

Growth hormone and epidermal growth factor together enhance amino acid transport systems B0,+ and A in remnant small intestine after massive enterectomy.

BACKGROUND: Sodium-dependent brush-border nutrient transport is decreased 2 weeks after massive enterectomy. This down-regulation is ameliorated by a 1-week infusion of parenteral growth hormone (GH) and epidermal growth factor (EGF) started 1 week after resection. We hypothesize that glutamine (GLN) transport will be enhanced by earlier and longer growth factor infusion, with differential effects on the Na(+)-dependent GLN transport systems A, B(0,+), and B(0)/ASCT2. MATERIALS AND METHODS: New Zealand White rabbits underwent 70% small bowel resection then immediately received parenteral EGF, GH, both EGF and GH, or neither for 2 weeks. Na(+)-dependent 3H-GLN uptake by jejunal and ileal brush-border membrane vesicles was measured and the contribution of systems A, B(0,+), and B(0) was then determined by competitive inhibition. Data were analyzed using one-way analysis of variance. RESULTS: In nonresected animals, the relative contribution of the systems was similar in jejunum (A 9%, B(0,+) 20%, and B(0) 71%) and ileum (A 13%, B(0,+) 27%, and B(0) 60%). Na(+)-dependent GLN uptake was reduced by one half in resected untreated controls, primarily because of decreased B(0) activity. EGF or GH alone did not affect Na(+)-dependent GLN transport, but, as a combination, there was increased uptake in the residual ileum and jejunum by 144% and 150%, respectively, over resected controls (P < 0.05). This was twice that achieved by delayed and shorter-duration combination treatment. This augmentation was a result of a 6.1-8.2-fold increase in system A as well as a 3.8-3.9-fold enhancement of system B(0,+) activity in remnant ileum and jejunum (P < 0.01). CONCLUSIONS: Parenteral EGF and GH, given in combination for 2 weeks immediately after massive enterectomy, synergistically enhance GLN uptake by systems A and B(0,+).

ATB0/ASCT2 expression in residual rabbit bowel is decreased after massive enterectomy and is restored by growth hormone treatment.

Two weeks after 70% enterectomy, glutamine (Gln) transport is downregulated in rabbit residual bowel due to a decrease in system B(0) activity. Providing epidermal growth factor (EGF) and growth hormone (GH) restores Gln transport by increasing systems A and B(0,+) activities. We hypothesized that changes in Na(+)-dependent broad-spectrum neutral amino acid transporter (ATB(0)/ASCT2) protein and mRNA expression correlate with system B(0) activity. New Zealand White rabbits underwent 70% jejunoileal resection or no resection. Resected rabbits immediately received parenteral EGF, GH, both, or neither agent for 2 wk. Tissues harvested from jejunum, ileum, and colon were subjected to Western and Northern blot analyses for ATB(0)/ASCT2 protein and mRNA. In all tissues, ATB(0)/ASCT2 mRNA was reduced by approximately 50% in resected rabbits compared with nonresected controls. Similar reductions in protein amount occurred in the ileum and cecum. None of the growth factor treatments restored ATB(0)/ASCT2 protein, but GH treatment increased ATB(0)/ASCT2 mRNA abundance 250% in the residual ileum. Because changes in the ATB(0)/ASCT2 protein amount paralleled those in the system B(0) activity in this model, it is likely that this is the protein responsible for this transport system. The increase in mRNA abundance in rabbits treated with GH for 2 wk may be a harbinger of subsequent increases in transporter protein and activity. Unlike reported upregulation of transporters in human colon after small bowel resection, ATB(0)/ASCT2 protein and mRNA expression in rabbit colon are decreased, suggesting different regulatory pathways.

Glutamine and KGF each regulate extracellular thiol/disulfide redox and enhance proliferation in Caco-2 cells.

Glutamine (Gln) and keratinocyte growth factor (KGF) each stimulate intestinal epithelial cell growth, but regulatory mechanisms are not well understood. We determined whether Gln and KGF alter intra- and extracellular thiol/disulfide redox pools in Caco-2 cells cultured in oxidizing or reducing cell medium and whether such redox variations are a determinant of proliferative responses to these agents. Cells were cultured over a physiological range of oxidizing to reducing extracellular thiol/disulfide redox (Eh) conditions, obtained by varying cysteine (Cys) and cystine (CySS) concentrations in cell medium. Cell proliferation was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. Gln (10 mmol/l) or KGF (10 microg/l) did not alter BrdU incorporation at reducing Eh (-131 to -150 mV), but significantly increased incorporation at more oxidizing Eh (Gln at 0 to -109 mV; KGF at -46 to -80 mV). Cellular glutathione/glutathione disulfide (GSH/GSSG) Eh was unaffected by Gln, KGF, or variations in extracellular Cys/CySS Eh. Control cells largely maintained extracellular Eh at initial values after 24 h (-36 to -136 mV). However, extracellular Eh shifted toward a narrow physiological range with Gln and KGF treatment (Gln -56 to -88 mV and KGF -76 to -92 mV, respectively; P < 0.05 vs. control). The results indicate that thiol/disulfide redox state in the extracellular milieu is an important determinant of Caco-2 cell proliferation induced by Gln and KGF, that this control is independent of intracellular GSH redox status, and that both Gln and KGF enhance the capability of Caco-2 cells to modulate extremes of extracellular redox.

Growth hormone and epidermal growth factor together enhance amino acid transport systems B(0,+) and A in remnant small intestine after massive enterectomy.

BACKGROUND: Sodium-dependent brush border nutrient transport is decreased 2 weeks after massive enterectomy. This downregulation is ameliorated by a 1-week infusion of parenteral growth hormone (GH) and epidermal growth factor (EGF) started 1 week after resection. We hypothesized that glutamine (GLN) transport would be enhanced by earlier and longer growth factor infusion, with differential effects on the Na(+)-dependent GLN transport systems A, B(0,+), and B0/ASCT2. MATERIALS AND METHODS: New Zealand White rabbits underwent 70% small bowel resection then immediately received parenteral EGF, GH, both, or neither for 2 weeks. Na(+)-dependent 3H-GLN uptake by jejunal and ileal brush-border membrane vesicles was measured and the contribution of systems A, B(0,+), and B0 then determined by competitive inhibition. Data were analyzed using one-way analysis of variance. RESULTS: In nonresected animals, the relative contribution of the systems was similar in jejunum (A, 9%, B(0,+), 20%; and B0, 71%) and ileum (A, 13%; B(0,+), 27%; and B0, 60%). Na(+)-dependent GLN uptake was reduced by half in resected, untreated controls, primarily because of decreased B(0) activity. EGF or GH alone did not affect Na(+)-dependent GLN transport, but as a combination, increased uptake in the residual ileum and jejunum by 144% and 150%, respectively, over resected controls (P<0.05). This was twice that achieved by delayed and shorter-duration combination treatment. This augmentation was due to a 6.1- to 8.2-fold increase in system A as well as a 3.8- to 3.9-fold enhancement of system B(0,+) activity in remnant ileum and jejunum (P<0.01). CONCLUSIONS: Parenteral EGF and GH, given in combination for 2 weeks immediately after massive enterectomy, synergistically enhance GLN uptake by systems A and B(0,+).