EFFECTS OF MODULATION OF TYROSINE PHOSPHORYLATION ON BRUSH-BORDER ENZYME-ACTIVITY IN HUMAN CACO-2 INTESTINAL EPITHELIAL-CELLS

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell type s and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphoryl ation in colonocyte differentiation, by using human colonic Caco-2 cel ls as a model and expression of the brush border enzymes alkaline phos phatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation marke rs. We studied three tyrosine kinase inhibitors with different modes o f action and specificities, viz., genistein, erbstatin analog (EA), an d tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanad ate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5- 75 mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stim ulation at 75mg/ml by 158.6+/- 17.5% and 228.6+/-37.1% of control valu es, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25 mM) similarly stimulated AKP and DPDD by 138.6/-6.6% and 131.8+/-1.5% of control values (n=12, P<0.001). Unexpectedl y, EA (0.1-10 mM) had the opposite effect, inhibiting AKP- and DPDD-sp ecific activity significantly at 10mM with a maximal 14.8+/-6.4% and 2 6.5+/-2.5% of control values (n=12, each P<0.001). Sodium orthovanadat e had a discordant effect on these two differentiation markers. Orthov anadate dose-dependently increased AKP to a maximal 188.5+/-16.1% of b asal activity at 1.5 mM but decreased DPDD activity at 1.5 mM to 47.2/-3.8% (n=9, P<0.001 each). The effects of each agent were preserved w hen proliferation was blocked with mitomycin C, suggesting that the mo dulation of phenotype by these agents was independent of any effects o f proliferation. The tyrosine phosphorylation of several phosphoprotei n bands was affected differently by these agents. In particular, the t yrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility th at at least two independent enzymes or pathways regulating tyrosine ph osphorylation modulate intestinal epithelial differentiation. Furtherm ore, tyrosine phosphorylation of the 70-kDa to 71-kDa phosphoprotein m ay be important in the intracellular signaling by which intestinal epi thelial cell differentiation is controlled.