Monica C. Chen

Effects of growth factors and trefoil peptides on migration and replication in primary oxyntic cultures.

Restitution, the lateral migration of cells over an intact basement membrane, maintains mucosal integrity. We studied the regulation of migration and proliferation of enzyme-dispersed canine oxyntic mucosa cells in primary culture. Confluent monolayers were wounded and cultured in serum-free medium, and cells migrating into the wound were counted. [3H]thymidine incorporation into DNA was studied using subconfluent cultures. Considerable migration occurred in untreated monolayers; however, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), two trefoil peptides, and interleukin (IL)-1beta further enhanced migration. The specific EGF receptor (EGFR) monoclonal antibody, MAb-528, inhibited both basal and TGF-alpha- or IL-1beta-stimulated migration, but not the response to trefoil peptide, bFGF, or IGF-I. Exogenous TGF-beta inhibited cell proliferation but did not alter migration. Immunoneutralization with anti-TGF-beta blocked the response to exogenous TGF-beta and produced a small enhancement of basal thymidine incorporation but did not attenuate basal or TGF-alpha-stimulated migration. In conclusion, endogenous EGFR ligands regulate proliferation and migration. TGF-beta inhibits mitogenesis; it did not upregulate migration in these cultures. Although bFGF, IGF-I, and IL-1beta enhance gastric epithelial migration, only IL-1beta acted in a TGF-alpha-dependent fashion.Restitution, the lateral migration of cells over an intact basement membrane, maintains mucosal integrity. We studied the regulation of migration and proliferation of enzyme-dispersed canine oxyntic mucosa cells in primary culture. Confluent monolayers were wounded and cultured in serum-free medium, and cells migrating into the wound were counted. [3H]thymidine incorporation into DNA was studied using subconfluent cultures. Considerable migration occurred in untreated monolayers; however, epidermal growth factor (EGF), transforming growth factor (TGF)-α, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), two trefoil peptides, and interleukin (IL)-1β further enhanced migration. The specific EGF receptor (EGFR) monoclonal antibody, MAb-528, inhibited both basal and TGF-α- or IL-1β-stimulated migration, but not the response to trefoil peptide, bFGF, or IGF-I. Exogenous TGF-β inhibited cell proliferation but did not alter migration. Immunoneutralization with anti-TGF-β blocked the response to exogenous TGF-β and produced a small enhancement of basal thymidine incorporation but did not attenuate basal or TGF-α-stimulated migration. In conclusion, endogenous EGFR ligands regulate proliferation and migration. TGF-β inhibits mitogenesis; it did not upregulate migration in these cultures. Although bFGF, IGF-I, and IL-1β enhance gastric epithelial migration, only IL-1β acted in a TGF-α-dependent fashion.

Modulation of gastric mucosal paracellular permeability by secretin: Role of src family tyrosine kinase

Background: Previous studies found that physiological concentrations of secretin increase transepithelial resistance (TER) in gastric mucosa in a fashion synergistic with EGF, indicating that spacially limited receptors and distinct signal transduction pathways are involved in the regulation of paracellular permeability. Pretreatment with PP2, a selective inhibitor of sm family of tyrosine kinase, significantly attenuated the increase in TER activated by secretio, but not by EGF, suggesting the src family kinases mediates secretin action on paracelloiar permeability. Aim and Methods: In order to identify the mechanism(s) by which secretio receptors lead to src family kinase activation, we examined the stimulation of src Idnsse activity measured by the autophosphorylation of Tyr416 and the inhibition of the overall profile of cellular protein tyrosine phosphorylation in primary canine gastric epithelial cell monoiayors by PP2(5/~M) after secretin (lnM) activation. Results: Primary gastric epithelial cell cultures showed consistent amount of phosphorylation of src Tyr416 at the basal levels and the stimulation by secretin only caused a maximal 2-fold activation of src activity after 10 to 15 min. However, this activation was not blocked by pretreatment of PP2. When total lysates were blotted with tyrosine phosphorylation antibody (4G10), several tyrosine phosphoryluted proteins activated by secretin were blocked by pretreatment with PP2 in a dose-dependent manner. The secretin-activated tyrosine phosphorylations were not inhibited by pretreatment with PP3(inactive src kinase inhibitor), nor with AG1478, a specific EGFR tyrosine kinase inhibitor, suggesting that these tyrosine pbosphofylated proteins serve either as src-family kinases or as substrates for these kinases. Conclusion: Our results showed that secretin activated specific tyrosine phosphorylated proteins via src-family tyrosine kinases. This finding suggests a novel function of src-family tyrosine kinases in regulating gastric paracelluiar permeability.