Inhibition of oxidant-induced barrier disruption and protein tyrosine phosphorylation in Caco-2 cell monolayers by epidermal growth factor

doi:10.1016/S0006-2952(98)00333-5

Biochemical Pharmacology

Volume 57, Issue 6, 15 March 1999, Pages 685-695

https://doi.org/10.1016/S0006-2952(98)00333-5 Get rights and content

Abstract

The effect of epidermal growth factor (EGF) on the H₂O₂-induced increase in paracellular permeability in Caco-2 and T-84 cell monolayers was evaluated to examine the role of EGF in intestinal mucosal protection from oxidative stress. Oxidative stress was induced by exposing cell monolayers to H₂O₂ or a mixture of xanthine oxidase + xanthine (XO + X). Paracellular permeability was assessed by measuring transepithelial electrical resistance (TER), sodium chloride dilution potential, and unidirectional flux of [³H]mannitol. H₂O₂ (0.1 to 5.0 mM) reduced TER and dilution potential and increased mannitol flux. Administration of EGF delayed H₂O₂ and XO + X-induced changes in TER, dilution potential, and [³H]mannitol flux. This protective effect of apically or basally administered EGF was concentration-related, with A₅₀ (95% confidence limits) values of 2.1 (1.17 to 4.34) and 6.0 (4.37 to 8.34) nM, respectively. The EGF-mediated protection was prevented by treatment of cell monolayers with genistein (10 μM), a tyrosine kinase inhibitor. H₂O₂ and XO + X also induced tyrosine phosphorylation of a number of proteins in Caco-2 and T-84 cell monolayers. EGF treatment inhibited the oxidant-induced tyrosine phosphorylation of proteins, particularly those with a molecular mass of 110–220 kDa. Treatment of Caco-2 cells with anti-transforming growth factor-α antibodies potentiated the H₂O₂-induced changes in TER, dilution potential, and mannitol flux. These studies demonstrated that an EGF receptor-mediated mechanism delays oxidant-induced disruption of the epithelial barrier function, possibly by suppressing the oxidant-induced tyrosine phosphorylation of proteins.

Section snippets

Cell culture

Caco-2 cells, originally obtained from Dr. Jeffrey Field, were maintained under standard cell culture conditions at 37° in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% (v/v) FBS. Cells were grown on polycarbonate membranes in Transwells (6.5 mm; Costar). Experiments were performed on day 12 or 13 after seeding cells onto Transwells (within passages No. 40–55). Under these conditions, confluent monolayers attained steady-state resistance to passive transepithelial ion flow, and

Paracellular permeability in Caco-2 cell monolayer

Baseline TER of Caco-2 cell monolayers (12- or 13-day post-seeding) varied from 300 to 450 Ω · cm². Administration of H₂O₂ to the basal surface at final concentrations of 0.1 to 5.0 mM reduced the TER in a time- and concentration-related manner (Fig. 1 ). Administration of EGF (30 nM) to the apical or basal buffer by itself produced no significant effect on baseline TER. However, EGF administered 60 min prior to H₂O₂ (5 mM) significantly inhibited the H₂O₂-induced decrease in TER (Fig. 2A ).

Discussion

The present study demonstrated that EGF delays oxidant-induced increase in paracellular permeability in Caco-2 and T-84 cell monolayers. Exposure to H₂O₂ (0.1 to 5.0 mM) reduced the TER of Caco-2 cell monolayers in a time- and concentration-related manner. This effect of H₂O₂ was associated with a decrease in sodium chloride dilution potential and an increase in unidirectional flux of [³H]mannitol. As described above, the concentration of H₂O₂ used is well within the pathophysiologic

Acknowledgements

This study was supported by a grant from the University Research Committee, Medical University of South Carolina.

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Hormones and Growth FactorsInhibition of oxidant-induced barrier disruption and protein tyrosine phosphorylation in Caco-2 cell monolayers by epidermal growth factor

Abstract

Section snippets

Cell culture

Paracellular permeability in Caco-2 cell monolayer

Discussion

Acknowledgements

Pharmacol Ther

Blood

J Biol Chem

Gastroenterology

Regul Pept

Eur J Pharmacol

Gastroenterology

Gastroenterology

Life Sci

J Biol Chem

Gastroenterology

Free Radic Biol Med

J Biol Chem

J Biol Chem

Exp Cell Res

Oxygen-derived speciesTheir relation to human disease and environmental stress

Environ Health Perspect

Free radicals and inflammationSuperoxide-dependent activation of a neutrophil chemotactic factor in plasma

Proc Natl Acad Sci USA

Oxidant-induced disruption of the intestinal epithelial barrier functionRole of protein tyrosine phosphorylation

Am J Physiol

Hormones and Growth Factors
Inhibition of oxidant-induced barrier disruption and protein tyrosine phosphorylation in Caco-2 cell monolayers by epidermal growth factor