CA2-DEPENDENT AND CA2+-INDEPENDENT REGULATION OF THE THYROID EPITHELIAL JUNCTION COMPLEX BY PROTEIN-KINASES()

The integrity of epithelial cell junctions is controlled by E-cadherin -mediated (Ca2+-dependent) cell-cell adhesion. In thyroid follicular c ells the dissociation of junctions induced by transfer to low Ca2+ med ium (Ca2+ switch) is prevented by thyrotropin acting via cyclic AMP/pr otein kinase A (cAMP/PKA) (Nilsson et al., Ear. J. Cell Biol. 56, 308- 318, 1991). In MDCK kidney epithelial cells protein kinase inhibitors elicit a similar response which, however, is cadherin-independent (Cit i, J. Cell Biol. 117, 169-178, 1992; Citi et al., J. Cell Sci. 107, 68 3-692, 1994). As such inhibitors also may interfere with PKA, we exami ned in a single cell type, filter-cultured pig thyrocytes, the effects and possible interactions of the cAMP/PKA agonist forskolin (or thyro tropin) and the kinase inhibitor H-7 in Ca2+ switch experiments. We fo und that the epithelial barrier dysfunction, comprising loss of transe pithelial resistance, increased transepithelial flux of [H-3]inulin an d redistribution of junction proteins (cadherin and ZO-1), which follo ws Ca2+ removal were inhibited by TSH, forskolin, and H-7. All agents were also able to induce recovery of resistance in low Ca2+. The maxim al recovery effects of forskolin and H-7 were additive when given simu ltaneous with Ca2+ chelator. In contrast, forskolin-induced recovery i nitiated 10 min after Ca2+ removal was antagonized by H-7. The protect ion of junctions by forskolin in low Ca2+ was rapidly abolished by lig ht trypsinization (0.001%), whereas the same concentration of trypsin had little or no effect on the corresponding action of H-7 or staurosp orine, another potent kinase inhibitor. In H-7-treated cells kept in l ow Ca2+, trypsin caused redistribution of ZO-1 from the plasma membran e to the cytoplasm while the transepithelial resistance remained high. Taken together, the data indicate that TSH via cAMP/PKA and the prote in kinase inhibitor H-7 reinforce the thyroid epithelial barrier under low Ca2+ conditions by distinct although interacting mechanisms. The high sensitivity to proteolysis in the absence of Ca2+ suggests that t he cAMP-regulated mechanism is cadherin-dependent. H-7 promotes or inh ibits the cAMP/PKA and the protein kinase inhibitor H-7 reinforce the thyroid epithelial barrier under Ca2+ conditions by distinct although interacting mechanisms. The high sensitivity to proteolysis in the abs ence of Ca2+ suggests that the cAMP-regulated mechanism is cadherin-de pendent. H-7 promotes or inhibits the cAMP/PKA-mediated recovery of tr ansepithelial resistance depending on the duration of the preceding lo w Ca2+ period. The trypsin-induced displacement of ZO-1 in H-7-treated cells in low Ca2+ suggests that the localization of ZO-1 to the tight junction is not necessary for the maintenance of junctional tightness . (C) 1996 Academic Press, Inc.