title: Ca2+-dependent and Ca2+-independent regulation of the thyroid epithelial junction complex by protein kinases.
abstract: The integrity of epithelial cell junctions is controlled by E-cadherin-mediated (Ca2+-dependent) cell-cell adhesion. In thyroid follicular cells the dissociation of junctions induced by transfer to low Ca2+ medium (Ca2+ switch) is prevented by thyrotropin acting via cyclic AMP/protein kinase A (cAMP/PKA) (Nilsson et al., Eur. J. Cell Biol. 56, 308-318, 1991). In MDCK kidney epithelial cells protein kinase inhibitors elicit a similar response which, however, is cadherin-independent (Citi, J. Cell Biol. 117,169-178,1992; Citi et al., J. Cell Sci. 107, 683-692, 1994). As such inhibitors also may interfere with PKA, we examined in a single cell type, filter-cultured pig thyrocytes, the effects and possible interactions of the cAMP/PKA agonist forskolin (or thyrotropin) and the kinase inhibitor H-7 in Ca2+ switch experiments. We found that the epithelial barrier dysfunction, comprising loss of transepithelial resistance, increased transepithelial flux of [3H]inulin and redistribution of junction proteins (cadherin and ZO-1), which follows Ca2+ removal were inhibited by TSH, forskolin, and H-7. All agents were also able to induce recovery of resistance in low Ca2+. The maximal recovery effects of forskolin and H-7 were additive when given simultaneous with Ca2+ chelator. In contrast, forskolin-induced recovery initiated 10 min after Ca2+ removal was antagonized by H-7. The protection of junctions by forskolin in low Ca2+ was rapidly abolished by light trypsinization (0.001%), whereas the same concentration of trypsin had little or no effect on the corresponding action of H-7 or staurosporine, another potent kinase inhibitor. In H-7-treated cells kept in low Ca2+, trypsin caused redistribution of ZO-1 from the plasma membrane to the cytoplasm while the transepithelial resistance remained high. Taken together, the data indicate that TSH via cAMP/PKA and the protein 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 the cAMP-regulated mechanism is cadherin-dependent. H-7 promotes or inhibits the cAMP/PKA-mediated recovery of transepithelial resistance depending on the duration of the preceding low 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.
authors.affiliation: Institute of Anatomy and Cell Biology, Göteborg University, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Nilsson
authors.forename: H
authors.initials: H
authors.lastname: Fagman
authors.forename: L E
authors.initials: LE
authors.lastname: Ericson
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004357:Drug Synergism; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium; D007546:Isoquinolines / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis; D010879:Piperazines / Q000494:pharmacology; D019311:Staurosporine; D013552:Swine; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D013972:Thyrotropin / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D014357:Trypsin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D000470:Alkaloids; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D008565:Membrane Proteins; D010750:Phosphoproteins; D010879:Piperazines; D062826:Zonula Occludens-1 Protein; D005576:Colforsin; D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D013972:Thyrotropin; D017868:Cyclic AMP-Dependent Protein Kinases; D014357:Trypsin; D019311:Staurosporine; D002118:Calcium