Differential Regulation of Endogenous Cadherin Expression in Madin-Darby Canine Kidney Cells by Cell-Cell Adhesion and Activation of β-Catenin Signaling*
- From the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5345
Abstract
Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin-associated cytoplasmic proteins α- and β-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997)J. Biol. Chem. 272, 29652–29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of β-catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active β-catenin mutants (ΔGSK, ΔN90, and ΔN131). In these cells, while levels of E-cadherin, α- and β-catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of ΔGSK-β-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post-translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype.
Footnotes
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↵* This work was supported in part by grants (to W. J. N.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Supported by a predoctoral fellowship from the Howard Hughes Medical Institute.
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↵§ Supported by a NATO fellowship from the Deutscher Akademischer Austauschdienst and an American Heart Association postdoctoral fellowship.
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↵¶ To whom correspondence should be addressed: Dept. of Molecular and Cellular Physiology, Beckman Center, Rm. B-121, Stanford University Medical Center, Stanford, CA 94305-5345. Tel.: 650-725-7596; Fax: 650-498-5286; E-mail: wjnelson@leland.stanford.edu.
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Published, JBC Papers in Press, March 27, 2000, DOI 10.1074/jbc.M000467200
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↵2 The data regarding the effects of proteasome and lysosomal inhibitors on cadherin-6 degradation are available as a figure upon request.
- Abbreviations:
- GSK
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glycogen synthase kinase
- MDCK
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Madin-Darby canine kidney
- PAGE
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polyacrylamide gel electrophoresis
- FPLC
-
fast protein liquid chromatography
- ALLN
-
N-acetyl-Leu-Leu-norleucinal
- TCF
-
T cell factor
- kb
-
kilobase(s)
- DMEM
-
Dulbecco's modified Eagle's medium
- Dox
-
doxycycline
- PVDF
-
polyvinylidene difluoride
- RT
-
reverse transcription
- PCR
-
polymerase chain reaction
- HRP
-
horseradish peroxidase
- TBS
-
Tris-buffered saline
- HSB
-
high stringency buffer
- CSK
-
cytoskeleton extraction buffer
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- Received January 19, 2000.
- Revision received March 27, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
