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Inactivation of glycogen‐synthase kinase 3 beta in Madin‐Darby Canine Kidney cells increases epithelial resistance
Author(s) -
Jouret Francois,
Rajendran Vanathy,
Caplan Michael J
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1152.13
Subject(s) - internalization , gsk 3 , extracellular , chemistry , cell culture , microbiology and biotechnology , kinase , glycogen synthase , tight junction , biology , phosphorylation , cell , biochemistry , genetics
Glycogen‐synthase kinase 3‐beta (GSK‐3β) is a ubiquitous serine‐threonine kinase involved in numerous biological processes. We have recently demonstrated that pharmacological inhibition of GSK‐3β activity leads to the assembly of tight junction (TJ) components in epithelial cells, even in the absence of extracellular Ca 2+ (Ca 2+ e ). Here, we generate and characterize a Madin‐Darby Canine Kidney cell line stably knocked‐down for the expression of the gene encoding GSK‐3β. At steady‐state, GSK‐3β inactivation is associated with an increased expression of ZO‐1 and occludin, at both the mRNA and protein levels. Transepithelial electrical resistance (TEER) is significantly higher in GSK‐3β KD (763.4 ± 54.5 ohm.cm 2 ) than in control (332.6 ± 38.6 ohm.cm 2 ) cells. Following Ca 2+ switch from low (5μM) to normal (1.8mM) Ca 2+ e , the relocation of ZO‐1 to cell‐cell contacts is significantly faster in GSK‐3β KD versus controls. Conversely, Ca 2+ e deprivation in control cells for 2 hours induces the internalization of the GSK‐3β substrate, β‐catenin, as well as a loss of measurable TEER. In GSK‐3β KD cells similarly exposed to low‐Ca 2+ e , β‐catenin stays localized predominantly at the basolateral membrane, and TEER remains measurable (197.1 ± 23.9 ohm.cm 2 ). These results support the conclusion that GSK‐3β activity modulates TJ assembly and stability in epithelial cells.